Mycobacterium Tuberculosis

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 162

Structural Studies of a Xyloglucan Endotransglycosylase from Populus tremula x tremuloides and Three Conserved Hypothetical Proteins from Mycobacterium tuberculosis

PATRIK JOHANSSON

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List of publications

This thesis consists of a comprehensive summary based on the following papers. In the text, the papers will be referred to by their roman numerals.

I Johansson P., Brumer H., Baumann M.J., Kallas Å.M., Henriks- son H., Denman S.E., Teeri T.T., Jones T.A. (2003). Crystalliza- tion and preliminary X-ray analysis of a xyloglucan endotransglycosylase from Populus tremula x tremuloides. Acta Cryst. D59, 535-537. II Johansson P., Brumer H., Baumann M.J., Kallas Å.M., Henriks- son H., Denman S.E., Teeri T.T., Jones T.A. (2004). Crystal structures of a poplar xyloglucan endotransglycosylase reveal details of transglycosylation acceptor binding. Plant Cell 16 (4), 874-886. III Johansson P., Unge T., Cronin A., Arand M., Bergfors T., Jones T.A., Mowbray S.L. (2005). Structure of an atypical epoxide hydrolase from Mycobacterium tuberculosis gives insights into its function. JMB 351 (5), 1048-1056. IV Castell A., Johansson P., Unge T., Jones T.A., Bäckbo K. (2005). Rv0216, a conserved hypothetical protein from Mycobacterium tuberculosis that is essential for bacterial survival during infection, has a double hotdog fold. Protein science 14 (7), 1850- 1862. V Johansson P., Castell A., Jones T.A., Bäckbro K. (2006). Struc- ture and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis. Submitted.

Articles I, II, III and IV have been reproduced with permission from the respective copyright holders. Additional publications

Henriksson L.M., Johansson P., Unge T., Mowbray S.L. (2004). X- ray structure of peptidyl-prolyl cis-trans isomerase A from Mycobac- terium tuberculosis. EJB 271 (20), 4107-4113.

Cronin A., Adamska M., Johansson P., Jones T.A., Mowbray S.L., Richter I., Unge T., Arand M. (2005). Characterisation of an epoxide hydrolase from Mycobacterium tuberculosis functionally related to mammalian cholesterol epoxide hydrolase. Naunyn-Schmiedeberg's Arch. Pharmacol. 371(1), R96.

Kallas Å.M., Piens K., Denman S.E., Henriksson H., Fäldt J., Jo- hansson P., Brumer H., Teeri T.T. (2005). Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris. Biochem. J. 390, 105-113. Contents

Inside the Matrix...... 11 Xyloglucan endotransglycosylases ...... 13 Structural studies of a Populus Xyloglucan Endotransglycosylase (Paper I & II)...... 16 Crystallization and structure determination...... 16 Structure...... 17 Active site...... 18 A XET-acceptor complex...... 19 XET donor binding...... 20 PttXET16A glycosylation...... 23 XET transglycosylation ...... 24 Conclusions and future perspectives...... 26 Phasing the unknown ...... 27 Mycobacterium tuberculosis...... 29 The M. tuberculosis genome ...... 30 Epoxide hydrolases ...... 30 Structure of M. tuberculosis Rv2740 (Paper III)...... 32 Crystallization and structure determination...... 32 Structure...... 33 Active site...... 34 Rv2740 activity and potential substrates ...... 35 Similarities to a human ChEH? ...... 36 Conclusions and future perspectives...... 37 Mtb fatty acid biosynthesis ...... 38 Fatty acid ȕ-oxidation...... 39 M. tuberculosis Rv0216 and Rv0130...... 41 Structure of M. tuberculosis Rv0216 (Paper IV)...... 41 Crystallization and structure determination...... 41 Structure...... 42 Putative active site ...... 43 Putative Rv0216 substrates?...... 45 Rv0216 a (de)hydratase? ...... 46 Related M. tuberculosis proteins ...... 47 Conclusions and future perspectives...... 49 Structure of M. tuberculosis Rv0130 (Paper V)...... 50 Crystallization and structure determination...... 50 Structure...... 50 Catalytic activity of Rv0130...... 52 Pockets, prolines and substrate specificity ...... 53 PHA biosynthesis ...... 54 Conclusions and future perspectives...... 56 Summary in Swedish ...... 57 Acknowledgements...... 59 References...... 61 Abbreviations

3RHDC R-3-hydroxydecanoyl-CoA 4HPC 4-hydroxyphenacyl-CoA ACP Acyl carrier protein ChEH Cholesterol 5,6-oxide hydrolase CoA Coenzyme A CYP Cytochrome P450 mono-oxygenase DHD Double hotdog EGXT Endo-xyloglucan transferase EH Epoxide hydrolase FabA E-hydroxyacyl-ACP dehydratase/isomerase FabD Malonyl-CoA:ACP transacylase FabG E-ketoacyl-ACP reductase FabI 2-trans-enoyl-ACP reductase FabM 2-trans, 3-cis-acyl-ACP isomerase FabZ E-hydroxyacyl-ACP dehydratase FAS Fatty acid synthase system GH Glycoside hydrolase Glc Glucose unit InhA 2-trans-enoyl-ACP reductase KasA / KasB E-ketoacyl-ACP synthase LEH Limonene epoxide hydrolase LTA4 Leukotriene A4 hydrolase MDR Multi drug resistance mEH Microsomal epoxide hydrolase Mtb Mycobacterium tuberculosis NCS Non-crystallographic symmetry PDB Protein Data Bank PHA Polyhydroxyalkanoate PttXET16A Populus tremula x tremuloides XET16A sEH Soluble epoxide hydrolase SeMet Selenomethionine SHD Single hotdog XET Xyloglucan endotransglycosylase XG Xyloglucan XTH Xyloglucan endotransglycosylase/hydrolase Xyl Xylose unit

Inside the Matrix

Xyloglucan endotransglycosylases

Cellulose is by far the most abundant polysaccharide in plants and accounts for 15-90% of the dry mass of primary and secondary cell walls, making it the most abundant biological compound on Earth (Coughlan and Folan, 1979). The cellulose polymer is composed of a non-substituted chain of D- glucose residues, linked by ȕ(1-4) glycosidic bonds. In plants, cellulose forms crystalline microfibrils that provide mechanical strength to the plant cell wall. The microfibrils, in turn are embedded in a complex matrix of hemicelluloses, lignin and structural proteins (Keegstra, 1973). One of the most abundant hemicelluloses of dicot plants is xyloglucan (XG). Xyloglu- can forms tight hydrogen bonds to cellulose and can thereby cross-link adjacent microfibrils (Valent, 1974). The xyloglucan backbone consists of ȕ(1-4)-linked glucosyl residues (Glc) substituted with Į(1-6)-linked xylose units (Xyl). The xylose branching pattern is quite regular, with three xylosylated glucose residues typically separated by one unbranched glucose residue. Some xylose residues are in turn further substituted by galactosyl and fucosyl-galactosyl groups (Carpita and Gibeaut, 1993). This extensive branching of the XG-backbone, prevents the xyloglucan molecules from forming crystalline microfibrils like cellulose. The plant cell wall contains a diverse array of enzymes able to modify different matrix polysaccharides. These include for example endoglucanases, that cleave the backbone of matrix sugars, and glycosidases that remove side chains, thus allowing more extensive interactions between adjacent polysaccharides (reviewed in (Fry, 1994)). When plant cell walls expand due to turgor pressure, the hemicellulose cross links need to be reconstructed (Fry, 1989). Endoglucanases and glycosidases that attack cellulose and hemicellulose are thought to be important for cell wall loosening (Rose and Bennett, 1999). However, such hydrolytic enzymes might undermine wall stability. In 1992, two independent research groups described the discovery of a new family of proteins capable of both cleavage and re-ligation of the xyloglucan backbone (Fry et al., 1992; Nishitani and Tominaga, 1992). These enzymes, originally called xyloglucan endotransglycosylases (XETs) or endoxyloglucan transferases (EGXT), catalyze like many endoglucanases cleavage of ȕ(1-4) bonds. However, they are in addition able to transfer the newly generated end to another long xyloglucan chain, thus allowing cell expansion

13 without compromising cell wall integrity (Purugganan et al., 1997; Smith and Fry, 1991; Xu et al., 1995). A few of the XET-like enzymes exhibit both xyloglucan endotransglycosylase and xyloglucan endohydrolase activity (Fanutti et al., 1993; Farkas et al., 1992). This apparent duality initiated Rose and coworkers (Rose et al., 2002) to suggest a new nomenclature for all XET-like proteins, combining them into a general xyloglucan endotransglycosylase / hydrolase family (XTH). The xyloglucan endohydrolase activity is in turn denoted XEH, whereas the xyloglucan endotransglycosylase activity is called XET. How- ever, in this thesis, XET will be used to denote an enzyme which is capable of performing xyloglucan transglycosylation. XET enzymes are usually subdivided into three to four major phylogenetic groups based on sequence similarity (Rose et al., 2002). This variation might reflect differences in expression, regulation, biochemical characteristics and thus in physiological function. Interestingly, many plant species encode a number of XET isoenzymes from different subfamilies (Dong et al., 2004). For example Arabidopsis thaliana encodes 33 XET-like genes, belonging to subfamilies 1-3 (Yokoyama and Nishitani, 2001). Several of these enzymes show differences in pH and temperature optima, as well as substrate specificity (Campbell and Braam, 1999). XETs have been shown to participate in cell wall expansion (Vissenberg et al., 2001; Vissenberg et al., 2000), adding new xyloglucan molecules into the cellulose-hemicellulose matrix. However, XET activity remains detectable in tissues that have ceased to grow (Pritchard et al., 1993), suggesting that XET enzymes are also involved in cell wall reconstruction and rein- forcement (Ito and Nishitani, 1999; Thompson et al., 1997). All presently known XET and XET-like sequences are members of glycoside hydrolase family 16 (GH16) (Henrissat and Davies, 1997). GH16 har- bors enzymes with widely different substrate specificity and is thought to have evolved from an ancestor with laminarinase activity (Barbeyron et al., 1998). In addition to XETs, family 16 encompasses 1,3-1,4 ȕ-glucanases (lichenases), keratan-sulfate-endogalactosidases, ț-carrageenases, ȕ- agarases, and 1,3- ȕ-glucanases (laminarinases). The members of family 16 are in turn clustered together with the cellobiohydrolases and the endoglucanases of family 7, to form glycoside hydrolase Clan B (Henrissat and Da- vies, 1997). Glycosidic bond cleavage by retaining glycosidases such as those in Clan B, generally takes place via a double-displacement reaction involving a pro- tonation step (glycosylation) and a deprotonation step (deglycosylation) (Koshland, 1953; White and Rose, 1997). In the first step, an active site carboxylate acts as a general acid and protonates the glycosidic oxygen. An- other nearby carboxylate simultaneously acts as a nucleophile, attacking the anomeric carbon of the sugar ring, thus forming a covalent glycosyl-enzyme intermediate (Vocadlo et al., 2001). In the second step, the general acid acts

14 as a general base and activates a water molecule, which in turn attacks the anomeric center and releases the sugar. In contrast to the strictly hydrolyzing enzymes of GH16 and GH7, XET deglycosylation of a bound oligosaccharide (donor molecule) includes deprotonation of another sugar moiety (termed the acceptor molecule). XETs normally use long XG-chains as donors, however, Fanutti and coworkers (Fanutti et al., 1996) showed that nasturtium seed XET could use short Glc8-based xyloglucan oligosaccharides as donor substrates. The smallest acceptor molecule in turn, seems to be a Glc3-based sugar, substituted by two xylose units on the first and the second glucosyl sugars on the non-reducing end (Lorences and Fry, 1993). Many XET isoenzymes prefer certain chemical features in both donor and acceptor molecules. Some are for example more active against fucosylated xyloglucan donors (Campbell and Braam, 1999), whereas a few might require ungalactosylated acceptors (Fanutti et al., 1996). Interestingly, some XETs show a much higher affinity towards high molecular weight (Mw) xyloglucan than to xyloglucan oligosaccharides (Purugganan et al., 1997).

15 Structural studies of a Populus Xyloglucan Endotransglycosylase (Paper I & II) Crystallization and structure determination Populus tremula x tremuloides XET16A (PttXET16A) was expressed in Pichia pastoris by Åsa Kallas, Hongbin Henriksson and Harry Brumer at the Royal Institute of Technology, Stockholm (Kallas et al., 2005). Crystalliza- tion trials of PttXET16A using Crystal Screen reagent kits (Hampton Re- search), resulted in two crystal forms (Figure 1).

Figure 1. The hexagonal and rod-like crystal forms of P. tremula XET16A.

Diffraction data of the rod-like and the hexagonal crystal forms were collected at the Max Lab synchrotron (Lund, Sweden) beamline I711 and at the ESRF synchrotron (Grenoble, France) beamline ID14:4 to a resolution of 3.5 Å and 2.1 Å, respectively. Autoindexing with Denzo (Otwinowski and Mi- nor, 1997) indicated that both crystal forms contained a primitive hexagonal lattice. Scaling statistics and systematic absences subsequently suggested that the rod-like crystals belonged to space group P3121 / P3221, whereas the hexagonal crystals belonged to space group P63 (unit-cell parameters a = 98.6 Å, b = 98.6 Å, c = 98.5 Å and a = 188.7 Å, b = 188.7 Å, c = 46.1 Å, respectively). In addition, four complete datasets of hexagonal crystals, soaked with various heavy atom solutions, were collected at Max Lab I711 and ESRF ID14:4 to resolutions ranging from 3 - 4 Å. One of these datasets, from a crystal soaked with 5mM KAuCl4, was found to exhibit a significant anomalous signal. Since this potential derivative-data was found to be almost isomorphous to the native, both anomalous and isomorphous difference Pat- terson maps were used to locate potential heavy atom sites. Three Au- positions were identified using the RSPS program (Knight, 2000) and subsequently cross-validated with MLPHARE (Otwinowski, 1991). Initial SIRAS phases were calculated by SHARP (de la Fortelle and Bricogne, 1997) to a resolution of 3.5 Å. The phases were in turn extended stepwise to a final resolution of 2.1 Å, using solvent flattening combined with twofold averaging in DM (Cowtan and Main, 1998).

16 The initial PttXET16A model was built interactively with O (Jones, 2004) and refined with the CNS package (Brunger et al., 1998), using a 4000 K simulated annealing procedure with strict non-crystallographic symmetry (NCS) constraints. Refinement was performed in REFMAC5 (Murshudov, 1997), using decreasing NCS-constraints. Water molecules were located using a combination of CNS Water-Pick (Brunger et al., 1998) and O, in peaks (>2ı) of the |2Fobs|-|Fcalc| electron density map. The refined XET structure was in turn used to solve the structure of XET in complex with a xyloglucan-derived nonasaccharide XLLG as well as two substrate analogues (Figure 2), by means of molecular replacement. Final phasing and refinement statistics are given in Paper II, Table 1. The PttXET16A structure and the PttXET16A-XLLG-complex have been deposited at the Protein Data Bank with PDB-codes 1UN1 and 1UMZ, respectively.

Figure 2. The XG-derived sugars used. (A) XLLG (B) XLLG-CNP (C) XLLG-NH2

Structure The PttXET16A molecule is dominated by two highly curved antiparallel ȕ- sheets, packing to form a globular jellyroll fold (Figure 3). The curvature of the upper ȕ-sheet creates a 35 Å long cleft, lined with aromatic residues. As expected, the overall XET structure is quite similar to the other known

17 members of the GH16 family. The PttXET16A ȕ-sandwich also shares remote similarity to both the cellobiohydrolases and the endoglucanases of glycoside hydrolase family 7. However, in contrast to the all-ȕ 1,3-1,4 ȕ- glucanases, the ȕ-agarases, the ț-carrageenases and the GH7 enzymes, the PttXET16A fold is completed by a C-terminal Į-helix. The C-terminal helix is in turn followed by an additional ȕ-strand, which seems to be conserved in all XET-like sequences. This ȕ-strand extends the potential substrate cleft and gives the upper sheet 8 strands compared to 7 in the lower. As in the GH16 / GH7 enzymes with known 3D-structures, the shape of the potential XET substrate binding cleft is not only governed by the makeup of the upper ȕ-strands, but also by the numerous loops and ȕ-turns that con- nect the two sheets. In PttXET16A the extended connection between strand ȕ13 and ȕ14, folds onto the concave sheet, constricting the width of the cleft by almost 10 Å. This makes the PttXET16A acceptor binding site (positive subsite identifiers, using the nomenclature of (Davies et al., 1997)) more closed compared to the ț-carrageenases and the ȕ-agarases, but more open compared to the 1,3-1,4 ȕ-glucanases. The 1,3-1,4 ȕ-glucanases, the ț-carrageenases and the ȕ-agarases feature an extended loop between ȕ2 and ȕ3, stretching into the donor side of the sugar-binding cleft (negative subsites). This long entrance loop is completely missing in XETs, exposing ȕ4 and parts of ȕ13 for substrate binding, thus making the XET donor binding subsites dramatically more open compared to the other known GH16 enzymes.

Figure 3. Overall structure and topology of PttXET16A.

Active site Despite substantial variations in sequence, substrate specificity and net reaction, the catalytic sites of XETs, 1,3-1,4 ȕ-glucanases, ȕ-agarases and ț- carrageenases of GH16 are surprisingly well-conserved (Figure 4AB). The putative PttXET16A catalytic nucleophile E85 is positioned 5.7 Å from the

18 putative general acid / base E89, consistent with a retaining glycoside hydrolase. As in the other GH16 enzymes, the potential nucleophile is coordinated by a low-barrier hydrogen bond to an intermediate aspartate side-chain (D87). In PttXET16A, a histidine residue (H83) is positioned at hydrogen bonding distance from the nucleophile. However, in the 1,3-1,4 ȕ-glucanases and the ȕ-agarases this position is occupied by a tryptophan side-chain, whereas in the ț-carrageenases by a tyrosine. Interestingly, this His- nucleophile interaction of PttXET16A seems to be conserved in most of the known XET sequences. The general acid / base in turn, does not form any hydrogen bonds with protein atoms. Consequently, the strands ȕ6 and ȕ8, flanking the PttXET16A active site, are not interacting with any of the catalytic residues and exhibit substantial variation within GH16.

A XET-acceptor complex In order to gain some clues about the nature of the XET transglycosylation reaction and how XETs are able to bind such a heavily branched substrate as xyloglucan, PttXET16A was co-crystallized with a xyloglucan-derived nonasaccharide XLLG (Figure 2A). The XET-XLLG complex structure was solved by molecular replacement to a resolution of 1.8 Å. Initial electron- density maps of the potential PttXET16A binding cleft displayed three glycosyl units of the XLLG-backbone, branched by two xylose rings. Subse- quent refinement using Refmac5 (Murshudov, 1997), revealed an additional galactose sugar, linked from the second xylose. The six sugars of the XLLG nonasaccharide wedge into the acceptor binding subsites of the protein to form an extensive number of interactions (Figure 4CD). In particular, the +1 glucose ring (Glc1) makes a close contact with the proposed acid / base E89, similar to the +1 glucose observed in both a family 7 endoglucanase substrate-analog complex (PDB-code 1OVW, (Sulzenbacher et al., 1996)) and in a Cel7A cellobiohydrolase-cellobiose complex (PDB-code 7CEL, (Divne et al., 1998)). However, compared to both family 7 structures, the XET-XLLG complex lacks a hydrogen bond between the Glc1 O3-hydroxyl and the carboxylate separating the two catalytic residues (D87). A water-mediated hydrogen bond is instead formed from the O4-hydroxyl to the putative nucleophile, mimicking the ȕ(1-4) linkage to an absent donor oligosaccharide. The binding of the rest of the branched sugar reveals a very favorable acceptor binding site, including a large number of hydrogen bonds and stack- ing interactions. Most importantly, the first and second glucose rings of the acceptor molecule are coordinated by the side-chain of W179, situated on the overhanging loop between strand ȕ13 and ȕ14. The indole ring of W179 also interacts with the first xylose ring (Xyl1), thus forming three sugar binding sites. The second xylose and the galactose ring are in turn coordinated by

19 Y250 and R258 on the XET-specific eight strand, explaining the differing topology in XETs compared to the other GH16 enzymes.

Figure 4. (A) and (B) Despite the differing enzymatic characteristics, as well as a ȕ- bulge present in the ȕ-agarases and the ț-carrageenases, the GH16 active site is surprisingly well-conserved. (C) and (D) The XLLG sugar, bound in the +1, +2 and +3 subsites of PttXET16A, underlines the importance of W179 and the long overhanging loop between strand ȕ13 and ȕ14. Contouring at 0.4 e/Å3.

XET donor binding Since no density for any sugars bound in the PttXET16A donor subsites could be seen in the XLLG complex, PttXET16A was co-crystallized with a potential inhibitor, xyloglucan amino-alditol XLLG-NH2 (Figure 2C). How- ever, the XLLG-NH2 substrate analogue exhibited an almost exact binding to the XET enzyme as the clean XLLG oligosaccharide, with three substituted glucose residues visible in electron density maps at subsites +1 to +3.

20 In the absence of a XET-xyloglucan donor complex, PttXET16A was compared to a number of enzyme-substrate complex structures from GH family 7 (Divne et al., 1998; Sulzenbacher et al., 1996), including a mosaic model of a cellulose nanomer spanning the catalytic center of a Trichoderma reesei cellobiohydrolase from subsites -7 to +2. In addition, PttXET16A was compared to the structure of a Bacillus macerans GH16 1,3-1,4 ȕ-glucanase in complex with a ȕ-glucan tetrasaccharide bound in the sites -4 to -1, which recently was deposited in the Protein Databank (PDB-code 1U0A, Gaiser et al., unpublished). A superposition of the active sites of the GH7 structures and the XET-XLLG complex, results in a decent overlap of the +1 glucose residue, the XLLG-Glc1 being slightly further away from the proposed nucleophile than its cellulose counterpart (Figure 5). Like in the GH7 enzymes and many other ȕ-glycosyl hydrolases, the -1 sugar of the 1,3-1,4 ȕ- glucanase is distorted. Due to the local similarity of PttXET16A to these enzymes, the distorted ring is readily fitted into the XET active site. The 6- hydroxyl group of the modeled -1 sugar interacts closely with several protein side-chains. Since this sugar cannot be further derivatized at 6-OH, this pro- vides an explanation to why XET enzymes preferentially cleaves after non- branched glucose units (Fry et al., 1992).

Figure 5. The XET-XLLG complex superimposed on a GH7 sugar-enzyme complex

The -2 subsites of the 1,3-1,4 ȕ-glucanases, the ȕ-agarases and the ț- carrageenases are located on a long loop partially covering the entrance of the substrate-binding cleft. Since this loop has no counterpart in the PttXET16A structure or in the known XET-like sequences, the -2 XET subsite cannot be determined based on the ȕ-glucanase complex (Figure 6). However, the family 7 enzymes are also lacking the entrance loop of the known GH16 structures. Curiously, the -2 sugar of the T. reesei cellobiohydrolase complex can be easily fitted to interact with the side-chains of W174 and S172 of the XET structure (Figure 5). Although the more distant donor

21 subsites are not as easily predicted, it is clear that XET donor-binding is more reminiscent of the substrate binding of endoglucanases and cellobiohydrolases than to the known family 16 enzymes.

Figure 6. PttXET16A (grey surface) features a significantly more open donor binding site than the other known GH16 enzymes (dark grey) due to differences in the loop between strand ȕ2 and ȕ3. The XLLG sugar, bound in the acceptor site of PttXET16A has been included for reference.

22 PttXET16A glycosylation Most of the XET subgroup 1-4 isoenzymes, including PttXET16A, feature at least one conserved N-X-S/T glycosylation site. Previous studies have indicated that the state of glycosylation might be of significant importance to enzyme activity. Removal of the N-linked glycosylation of subgroup 2 Arabidopsis TCH4 XET by enzymatic treatment, resulted in the near-total loss of transglycosylase activity (Campbell and Braam, 1998; Campbell and Braam, 1999). However, more recent studies of Brassica oleracea var. bo- trytis BobXET16A, indicate that glycosylation might not be required for any of the XET subgroups, accounting previous results to normal temperature and pH instability (Henriksson et al., 2003). PttXET16A also retains full enzymatic activity upon deglycosylation using Endo H (Kallas et al., 2005). Strong electron density for two acetylglucoseamine rings and one mannose ring, attached to asparagine 93, was found in the experimentally phased maps of native PttXET16A (Figure 7A). The glycosylation sugar makes close interactions with the overhanging loop between ȕ13 and ȕ14. Since this loop is responsible for the formation of the +1 +2 & +3 subsites, removal of the N-linked oligosaccharide might potentially lead to a destabili- zation of the acceptor site. However, in PttXET16A the loop is also stabilized by a complex network of consecutive salt bridges (Figure 7B). Several of these electrostatic interactions seem to be conserved in BobXET16A but not in the Arabidopsis TCH4 enzyme, suggesting that they might be the rea- son for the retained activity of PttXET16A and BobXET16A after deglycosylation.

Figure 7. (A) The PttXET16A glycosylation sugar interacts with the overhanging acceptor loop, thus linking it to strand ȕ7 and ȕ8. (B) The acceptor loop is further stabilized by three bidentate salt bridges, only conserved in some of the XET-like sequences.

23 XET transglycosylation The catalytic mechanism of XETs and other retaining ȕ-glycosyl hydrolases has been proposed to include a covalent saccharide-enzyme intermediate and / or an oxocarbenium-ion-like intermediate (Imoto, 1972; Koshland, 1953). This short-lived intermediate is in turn hydrolyzed by a water molecule that has been activated by the residue acting as a general acid in the first step of the reaction. XETs, however, are believed to delay hydrolysis until an acceptor oligosaccharide diffuses into the active cleft and subsequently completes a transglycosylation reaction . Biochemical studies have indicated that PttXET16A is a true transglycosylase which possesses no detectable hydrolytic activity toward xyloglucan (Kallas et al., 2005). As such, water itself is unable to act as an acceptor in the breakdown of a putative xyloglucan glycosyl-enzyme intermediate. However, short XGO molecules and reducing-end derivatives do function as acceptors with PttXET16A when high Mw XG is used as a donor, as has been observed for other XET enzymes (Sulova et al., 1995). To further elucidate the enzymatic characteristics of PttXET16A, Harry Brumer and coworkers at the Royal Institute of Technology, synthesized a novel xyloglucan oligosaccharide bearing a chromogenic aglycone, XLLG- CNP (Figure 2B), as a potential glycosyl donor substrate. However, PttXET16A was unable to use XLLG-CNP as a transglycosylation donor, and no release of the chromophoric 2-chloro-4-nitrophenylate group could be observed. In contrast, XLLG-CNP readily acted as an acceptor when high Mw xyloglucan was used as a donor (Paper II, Figure 5). One possibility for the failure of XLLG-CNP to act as a glycosyl donor might be a requirement for binding a longer xylogluco-oligosaccharide chain in the acceptor subsites to properly orient the phenolic glycosidic bond relative to the catalytic residues. Based on this reasoning, it is tempting to speculate that the general transglycosylation reaction might be rationalized in terms of the activation of the enzyme only when xyloglucan-derived sugars are bound in donor and acceptor sites. Binding of a sugar chain in both the donor and acceptor sites correctly positions the catalytic machinery to allow the formation of a glycosyl-enzyme intermediate. Diffusion of the newly formed non-reducing end from the active site then causes the enzyme to assume a catalytically inactive conformation which is stable to hydrolysis. Subsequent binding of a carbohydrate acceptor in the positive subsites re- stores the catalytically active conformation, thus allowing glycosyl transfer from the enzyme to occur (Figure 8).

24 Figure 8. The proposed XET transglycosylation reaction based on an activation / inactivation mechanism. Like other retaining glycoside hydrolases XETs are believed to act through general acid catalysis via an oxocarbenium ion-like transition state (‡). However, when the newly cut XG-sugar is released from the XET acceptor site, the catalytic machinery of the enzyme is inactivated (c), thereby extending the lifetime of the covalent intermediate. The catalytic XET machinery can subsequently be reactivated upon binding of a xyloglucan acceptor chain, enabling a transglycosylation reaction.

25 Conclusions and future perspectives Populus tremula x tremuloides XET16A represents the first 3D-structure of a xyloglucan endotransglycosylase, capable of cleaving and rejoining xyloglucan molecules. PttXET16A shares a conserved ȕ-jelly roll topology with the diverse glycoside hydrolase family 16, but features a unique substrate binding site, capable of recognizing a heavily branched xyloglucan sugar. The active site in turn, is surprisingly well-conserved within GH16, despite differences in substrate specificity and overall reaction. One of the few differing side-chains interacting with the catalytic machinery is His83. Since this residue is conserved in many XET enzymes, it might be an interesting target for site-directed mutagenesis. The XET-XLLG complex revealed a very favorable acceptor binding site, which is necessary but probably not sufficient for XET catalysis. Enzymatic studies suggest that the binding of xyloglucan sugars in both positive and negative subsites might be a requirement for the first, and possibly, the second step of transglycosylation. However, the conformation of active-site residues in the native structure and the XET-XLLG acceptor complex are essentially identical, and does not provide any direct clues on how such a putative activation/inactivation could be achieved. A XET-donor complex could potentially shed some light on this issue, and on the nature of the putative covalent enzyme-saccharide intermediate. However, substantial biochemical studies might still be required to elucidate the details of the transglycosylation reaction, and how such an intermediate can be stabilized in an aqueous solution.

26 Phasing the unknown

Mycobacterium tuberculosis

The Mycobacterium genus consists of a large number of subspecies, including the human pathogens Mycobacterium tuberculosis (Mtb) and Mycobac- terium leprae. Despite major efforts to combat tuberculosis (TB), the disease remains a severe public-health problem. Over the next 25 years, an estimate of 40 million people will die from TB (WHO, 2001). The gram-positive bacterium Mycobacterium tuberculosis was discovered by Koch in 1882. However, no effective treatment of tuberculosis was available until 1943, when the aminoglycoside streptomycin was introduced. A number of potent TB drugs were subsequently discovered in the 1950-60s, including isoniazid, rifampicin, and pyrazinamide, which are all still being used today. Even with these drugs the bacteria is very difficult to eliminate due to its tendency to hide in an inactive, latent state. The subsequent long treatment periods lead to poor drug compliance, resulting in the emergence of multi drug-resistant (MDR) bacterial strains, constantly undermining the activity of the classical drugs (reviewed in (Stewart et al., 2003)). In addition, the recent AIDS epi- demic has led to a resurgence of tuberculosis and creates an urgent need for the development of new antimycobacterial agents. M. tuberculosis primarily infects macrophages, which normally represent a first defense against microbial invasion. The bacterium is protected by a thick waxy cell wall which helps it survive in the hostile environment of the macrophage's phagocytic vacuole (recently reviewed in (Nguyen and Pieters, 2005)). In addition, Mtb interfere directly with the macrophage, inhibiting the acidification and maturation of the phagosome as well as expresses a range of enzymes that detoxify oxidative radicals (Koul et al., 2004; Sturgill- Koszycki et al., 1994). The infected macrophages in turn, form so-called granulomas together with T-lymphocytes to try to confine the intruder (Flynn and Chan, 2003). Granuloma formation is usually sufficient to limit a normal bacterial infection. However, since M. tuberculosis is able to survive within the oxygen and nutrient-depleted macrophages, it can hide from the rest of the immune system and stay latent inside the host for long periods of time (O'Regan and Joyce-Brady, 2001). The lipid-rich cell wall of M. tuberculosis features two layers; a normal inner membrane followed by a peptidoglycan layer, covalently attached to arabinogalactan (Brennan, 2003). The arabinogalactan molecules are in turn connected to long mycolic acids. The Mtb cell envelope exhibits very poor permeability and form an efficient barrier against hydrophilic compounds,

29 likely to account for some of the unusual resistance of mycobacteria to anti- biotics and other toxic substances (Brennan and Nikaido, 1995).

The M. tuberculosis genome Recently, the genomes of several mycobacterial species have been se- quenced. The complete M. tuberculosis genome (Cole et al., 1998), contains ~4000 open reading frames. Intriguingly, about 50% of the genes show signs of recent gene duplication or domain shuffling (Tekaia et al., 1999), support- ing the hypothesis that Mtb is a relatively young bacteria in evolutionary terms (Kapur et al., 1994; Sreevatsan et al., 1997). Cole and coworkers (Cole et al., 1998) were able to predict the function of 40% of the protein-coding genes with some level of confidence. A second group of 44% showed similarity to putative proteins of other organisms and were subsequently classified as conserved hypothetical proteins of unknown function. The remaining 16% did not show any similarity to any known gene or protein. The M. tuberculosis genome seems to be unusually abundant in genes involved in regulation of gene expression, fatty acid metabolism and transport of metabolites. For example, Mtb seems to have over 250 proteins involved in fatty acid synthesis and degradation. In comparison, E. coli has only about 50 such enzymes (Cole et al., 1998), potentially reflecting the complex cell wall and the harsh life style of M. tuberculosis. Compared to the 4000 open reading frames of the Mtb genome, M. leprae seem to have only about 1600 active genes (Cole et al., 2001). Half of the M. leprae genome is composed of non-coding pseudo-genes; genes that have coding orthologs in M. tuberculosis and other mycobacteria. This apparent genome reduction seems to have left a minimal set of functional genes, giving hints about genes required for mycobacterial survival. In addition, several specific experimental methods have been developed to identify essential M. tuberculosis genes under various in vitro conditions mimicking infection (Betts et al., 2002; Sassetti et al., 2001; Sherman et al., 1995). More recently, Sassetti and coworkers used a so-called transposon site hybridization method to determine genes important for in vitro (Sassetti et al., 2003) and in vivo growth (Sassetti and Rubin, 2003). Even though these whole-genome approaches show surprisingly little correlation (Kendall et al., 2004), they give a starting point for selecting targets aimed for more detailed studies.

Epoxide hydrolases An epoxide is a highly reactive organic three-membered oxygen compound that arises from the metabolism of lipophilic substrates via e.g. the cytochrome P450-dependent mono-oxygenase system (Fretland and Omiecinski,

30 2000). These oxirane derivatives can in turn be degraded by so-called epoxide hydrolases (EHs). The epoxide hydrolases seem to have three main functions; detoxification, synthesis of signaling molecules and catabolism of specific carbon sources. Eight different classes of epoxide hydrolases have been described in pro- and eukaryotes, including two classes of soluble EH (sEH), one class of microsomal EH (mEH), as well as the smaller classes of cholesterol 5,6-oxide hydrolase (ChEH), hepoxilin A3 hydrolase, leukotriene A4 hydrolase (LTA4), limonene EH (LEH) and juvenile hormone EH (recently reviewed in (Newman et al., 2005)). Both the soluble and the microsomal epoxide hydrolases belong to a broad family of 35-40 kDa Į/ȕ hydrolase folded proteins that act via a two-step catalytic mechanism. mEH is primarily involved in elimination of genotoxic epoxides generated by oxygenase enzymes, whereas sEH is involved both in detoxification and in processing of signaling molecules (Arand et al., 2003a). In contrast, the hepoxilin A3 hydrolase and the cholesterol 5,6-oxide hydrolase have not been characterized. The latter enzyme has attracted substantial interest due to its potential role in the regulation of vascular homeostasis (Hu et al., 1991; Mahfouz et al., 1996; Peng et al., 1991), but has thus far resisted purification and cloning (Newman et al., 2005). The leukotriene A4 hydrolase and the limonene EH differ significantly from the Į/ȕ-hydrolase folded enzymes both in structure and in function. The LTA4 is a 62 kDa, three-domain, met- alloprotein involved in inflammatory and allergic response (Thunnissen et al., 2001), whereas LEH is a small 16 kDa bacteria-specific enzyme related to degradation of monoterpenes (Arand et al., 2003b; Barbirato et al., 1998). The M. tuberculosis genome encodes at least 20 cytochrome P450- dependent mono-oxygenases (CYP) (Cole et al., 1998; Souter et al., 2000), an unprecedented number for a bacteria. In eukaryotes the P450-enzymes are often involved in xenobiotic degradation, whereas in bacteria they are mainly used in the degradation of organic compounds for metabolic fuel (Munro et al., 2003). M. tuberculosis also seems to contain an unusually large number of epoxide hydrolases (Tekaia et al., 1999). Nine Mtb proteins (Rv0134, Rv1124, Rv1938, Rv2214c, Rv3617, Rv3670, Rv1834, Rv3176c and Rv2740), several of which have been classified as conserved hypothetical proteins, were recently shown to have epoxide hydrolase activity (Arand et al., unpublished). Eight of these seem to belong to the Į/ȕ-hydrolase family, whereas the last protein of the group, Rv2740, is more similar to the limonene epoxide hydrolase. Notably, four of the eight Į/ȕ-epoxide hydrolases, as well as the Rv2740 are conserved in the minimal M. leprae genome.

31 Structure of M. tuberculosis Rv2740 (Paper III) Crystallization and structure determination Native Rv2740 was cloned, expressed and purified by Terese Bergfors. Ini- tial screening for native crystallization conditions was performed by the sparse matrix CoreScreen (Page and Stevens, 2004) using vapor diffusion. Diffraction data of the most promising crystals were collected at MAX Lab, beamline I711, and at ESRF, beamline ID14:1, to a resolution of 3 Å. The data were processed and reduced using the Denzo / Scalepack suite (Otwinowski and Minor, 1997) and was found to belong to space group P3121 / P3221, with unit cell parameters a=80.6 Å, b=80.6 Å and c=118.7 Å.

Figure 9. Crystals of M. tuberculosis Rv2740 in sizes from 20–70 ȝm.

For phasing purposes, twelve drops containing native crystals were soaked with different heavy atom solutions, including Ag, Au, Cd, Ho, Pb and various Hg compounds, in concentrations between 0.2 and 10 mM. SAD and MAD data of nine of these heavy atom soaked crystals were collected at beamlines I711, MAX Lab, ESRF ID14:4 and ESRF ID29. Several of the mercury datasets exhibited a strong anomalous signal, and the Hg- substructure seemed to be readily solved by SHELXD (Schneider and Shel- drick, 2002). However, neither SHARP (de la Fortelle and Bricogne, 1997), SHELXD or SOLVE (Terwilliger and Berendzen, 1999) were able to properly refine the heavy atom positions, and the subsequent density-modified maps could not be interpreted. To overcome these problems, selenomethionyl-substituted (SeMet) Rv2740-protein was prepared by Torsten Unge. The SeMet Rv2740 could be crystallized under similar conditions as the native protein and a near-edge SAD dataset was subsequently collected at ESRF beamline ID14:2 to a resolution of 2.5 Å. Six selenium atoms were identified using the RSPS program (Knight, 2000) and refined with SHARP (de la Fortelle and Bricogne, 1997). Based on density modification results, the correct space group was determined to be P3221. The NCS operators relating the three molecules of the asymmetric unit were identified using O (Jones, 2004), refined with Imp (Jones, 1992) and used for averaging in DM (Cowtan and Main, 1998). The

32 Rv2740 structure was built in O and refined in the CNS package (Brunger et al., 1998) as well as in REFMAC5 (Murshudov, 1997) using a decreasing amount of NCS constraints. Final phasing and refinement statistics are given in Paper III, Table 2. The Rv2740 structure and structure factors have been deposited at the Protein Data Bank with PDB-code 2BNG.

Structure The Rv2740 monomer is composed of a 6-stranded mixed ȕ-sheet folded on top of three Į-helices to form a cone-shaped molecule of ~35×25×25 Å (Figure 10). The high curvature of the ȕ-sheet in combination with a fourth N-terminal helix that packs onto the edge of the sheet, create a deep cavity of more than 300 Å3 (CASTp server, Connolly's volume, (Naghibzadeh et al., 2001)). The Cystatin-like Į+ȕ fold of Rv2740 is shared by a number of proteins with differing functions, including the limonene 1,2-epoxide hydrolase of R. erythropolis (Paper III, Table 3). All of these proteins exhibit a similar distinctive deep pocket, formed by the large curved sheet. However, in some of the structures, including a scytalone dehydratase (Chen et al., 1998) and a protein of unknown function, Bal32a (Robinson et al., 2005), the opening of the cavity is closed by an extended ȕ-turn or by the N-terminus.

Figure 10. (A) The two Rv2740 monomers form a dimer related by a nearly prefect 2-fold axis (B) Both the dimer and the Rv2740 monomer are stabilized by a large number of salt bridges, the monomer encompassing 14 arginines in 149 residues.

As in many of the similar structures, two Rv2740 molecules form a dimer. The highly complementary ȕ-sheets of the two subunits pack perpen- dicularly, burying a total accessible surface area of over 2000 Å2. In addition to the hydrophobic interactions of the dimer interface, the subunit-formation is further stabilized by a complex network of 4+4 connected salt bridges (Figure 10B). Both Rv2740 and the unknown function Bal32a seem to fea-

33 ture a reduced cysteine bridge situated in the dimer interface. In Bal32a, CD- measurements indicate that a disulphide bond indeed exists in the absence of reducing agents (Robinson et al., 2005).

Figure 11. (A) Rv2740 active site. (B) The Rv2740 (blue) features a significantly larger substrate binding cavity than the Rhodococcus LEH (red). (C) and (D) Den- sity for a large endogenous ligand, bound close to the active site, was found in maps of all Rv2740 datasets. Contouring at 0.4 e/Å3.

Active site The deep cavity of Rv2740 is principally lined with hydrophobic side- chains. However, a band of polar residues stretch down from the rim of the pocket towards the bottom, including the side-chains of D93, R91 and D122. A superposition of the R. erythropolis limonene 1,2-epoxide hydrolase onto Rv2740, reveals that these three residues overlap exactly with the catalytic triad of LEH (Arand et al., 2003b). In addition, the side-chains of Y46 and N48 fit the LEH structure, creating a well-conserved active site (Figure

34 11A). The limonene epoxide hydrolase is thought to act via a single step push-pull mechanism, whereby the counterpart of Rv2740 D93 donate a proton to the oxirane ring. The counterpart of D122 abstracts a proton from a catalytic water molecule, which in turn attacks the epoxide carbon. The intermediate residue, R91, positions the two aspartates and stabilizes the negative charge, flipping from D122 to D93 (Arand et al., 2003b) (Paper III, Fig- ure 1). This mechanism was recently verified by a density functional theory method (Hopmann et al., 2005), and is likely to be used by Rv2740 as well. In contrast to the highly conserved active site, the surrounding cavities of the two proteins share very little similarity. Slight changes in the orientation of two of the helices together with differences in sequence, makes the Rv2740-pocket substantially wider and deeper than its LEH counterpart (Figure 11B). The experimentally phased maps of Rv2740 contained strong electron density for an unexpected ligand, bound in the deep cavity in all of the three molecules of the asymmetric unit (Figure 11CD). This density seems to be consistent with a substituted ring-structure with an aliphatic extension. How- ever, the relatively low 2.5 Å resolution of the Rv2740 data, makes it hard to distinguish the chemical nature of this unknown compound. A similar active site density was found in all of the nine datasets of the heavy-atom soaked crystals. Curiously, both the LEH and the BAL32a structures also contained endogenous ligands (Arand et al., 2003b; Robinson et al., 2005), albeit of different sizes and shapes.

Rv2740 activity and potential substrates Due to the similarity of Rv2740 to the limonene 1,2-epoxide hydrolase, Rv2740 was assayed against an array of various epoxides. The enzyme was found to be active on three structurally very different compounds; styrene 7,8-oxide, cis-9,10-epoxystearic acid and cholesterol 5,6-epoxide (Paper III, Figure 1)(Paper III, Table 1). Although none of the three tested epoxides are likely to be used by Rv2740 in vivo, they give hints about the nature of true physiological Rv2740-substrates. In contrast to the small limonene-1,2- epoxides of LEH, Rv2740 is likely to act on bulky fatty acid or steroid derivatives, consistent with both the hydrophobic character of the large putative substrate binding site, as well as the size and shape of the endogenous ligand. Like in the LEH case, the epoxide hydrolase activity could be inhibited by the antiepileptic drug valpromide (2-propyl pentanamide) with a KI of about 100 ȝM. Attempts were made to solve an enzyme-valpromide complex, but the electron-density maps of inhibitor-soaked crystals did not show unambi- guous density for a single compound in the active site. This might possibly be due to competitive binding of the endogenous ligand. However, valpromide was found to weakly inhibit at least six of the M. tuberculosis Į/ȕ-

35 epoxide hydrolases (Arand et al., unpublished), indicating the potential of multiple target EH inhibition. It has been suggested that the many M. tuberculosis P450-dependent CYP enzymes are just a remnant from a putative soil-living Mtb-ancestor, since only one of them, CYP140, is conserved in the M. leprae genome (Cole, 2002a). However, several azole drugs, which are known CYP51 inhibitors, severely inhibit mycobacterial growth, suggesting a key function for these enzymes (McLean et al., 2002). Even though little is known about the physiological role of the Mtb P450-dependent enzymes, several of them seem to be involved in sterol modification (Aoyama et al., 1998; Bellamine et al., 1999), indicating the presence of such compounds in mycobacteria. Epoxides of sterol derivatives could in turn potentially be exploited by enzymes like Rv2740.

Similarities to a human ChEH? Since no cholesterol epoxide hydrolase has been purified to homogeneity (Newman et al., 2005), Paper III represents the first detailed characterization of an enzyme with such an activity. Although the human ChEH is thought to be membrane-associated, previous studies have suggested a relationship between LEH and ChEH (Arand et al., 2003b). ChEH is much smaller than the Į/ȕ-hydrolase folded enzymes (Watabe et al., 1986) and does not seem to employ the classical epoxide hydrolase two-step reaction. The human enzyme is also like LEH and Rv2740 able to hydrolyze trisubstituted epoxides and shares similar stereoselective properties. It is possible that ChEH shares both structural and mechanistic similarities with the LEH-like proteins. However, no convincing homolog to any of the nine Rv2740 / LEH-like proteins (Paper III, Figure 4) could be found in the human genome using WU-Blast 2.0 (http://blast.wustl.edu, (Lopez et al., 2003)).

36 Conclusions and future perspectives The Rv2740 monomer forms a Cystatin-like Į+ȕ fold similar to several known structures, including a limonene 1,2-epoxide hydrolase from R. erythropolis. Rv2740 shares not only its structure with LEH, but also epoxide hydrolase activity. However, Rv2740 exhibits substantially different substrate specificity compared to the Rhodococcus enzyme. The substrate specificity, the large binding pocket, as well as the size and shape of the endogenous ligand found in the structure, suggests that Rv2740 might be involved in metabolism of fatty acid or sterol epoxides. Such compounds might in turn be derived from P450-dependent degradation of exogenous organic compounds taken up by the mycobacteria. Interestingly, Rv2740 and several of the Mtb Į/ȕ-epoxide hydrolases can be inhibited by the already existing antiepileptic drug valpromide, thus opening a possibility for multiple target inhibition. Since the endogenous ligand could only partially be displaced by valpromide, this mysterious compound is also of interest for improving existing inhibitors. Mass spectroscopy studies, in collaboration with Michael Arand (University of Zürich), are currently underway to try to elucidate the nature of this unknown ligand.

37 Mtb fatty acid biosynthesis M. tuberculosis is able to produce a very diverse set of fatty acids using two distinct fatty acid synthase systems, FAS I and FAS II, as well as a number of auxiliary enzymes (recently reviewed in (Takayama et al., 2005)). The FAS I system consists of a large seven-domain protein that catalyzes all the various steps in the synthesis of short-chain fatty acids. The acyl-CoA esters produced by FAS I are subsequently transferred to an Mtb acyl carrier protein (ACP) by a malonyl-CoA:ACP transacylase and shuttled into the FAS II system. The fatty acid synthase II system in contrast, is composed of separate enzymes with similar functionalities to the domains of FAS I. Both FAS I and most of the FAS II subunits have been identified in the Mtb genome (Cole et al., 1998). However, two of the postulated fatty acid synthase system II enzymes are still missing; a ȕ-hydroxyacyl-ACP dehydratase (FabZ), involved in the normal FAS II cycle (Figure 12), and a 2-trans-enoyl-ACP isomerase (FabM), involved in the synthesis of unsaturated meroacids (Takayama et al., 2005).

Figure 12. The FAS II fatty acid synthase system of M. tuberculosis.

38 Based on remote similarity to two predicted ȕ-hydroxyacyl-ACP dehydratases, Takayama and coworkers (Takayama et al., 2005) recently suggested Rv0098 and Rv0130 to be potential candidates for the missing FabZ enzyme. However, only part of the classical dehydratase active site residues (Kimber et al., 2004; Leesong et al., 1996) seem to be conserved in Rv0098, whereas none are conserved in Rv0130. Since the fatty acid synthase systems are critical for M. tuberculosis survival, substantial efforts have been put into finding inhibitors to the different components of FAS I and FAS II (Duncan, 2000; Heath et al., 2001). Isoni- azid, one of the classical tuberculosis drugs, was in 1994 found to be involved in the inhibition of at least two enzymes in the fatty acid synthase system II, the enoyl-ACP reductase InhA (Banerjee et al., 1994) and the ȕ- ketoacyl-ACP synthase KasA (Figure 12) (Mdluli et al., 1998), leading to blockage of mycolic acid synthesis.

Fatty acid ȕ-oxidation Mtb is thought to absorb a variety of fatty acids from the host cell for energy metabolism (Barclay, 1989; Wheeler et al., 1990; Wheeler et al., 1991). In correlation, the M. tuberculosis genome was found to contain an unusually large number of genes potentially involved in fatty acid catabolism (Cole et al., 1998). In addition to the normal prokaryotic (aerobic) FadA / FadB multifunctional ȕ-oxidation enzymes encoded by Rv0859 and Rv0860, Mtb contains about a hundred genes predicted to catalyze all the individual steps of lipid degradation. The general fatty acids ȕ-oxidation requires four different enzyme- catalyzed reactions; desaturation, hydration, dehydrogenation and thiolation (Nunn, 1986). In contrast to eukaryotes, most bacteria use one or two simpli- fied pathways, including an S-hydroxyacyl-CoA intermediate (Figure 13). The apparent abundance of lipid degrading enzymes in M. tuberculosis, indicates that other ȕ-oxidation pathways might well exist. However, not much is known about the enzymes involved or how Mtb transports and oxidizes exogenous fatty acids.

39 Figure 13. . Schematic representation of the ȕ-oxidation spiral. Bacteria generally use the S-specific ȕ-oxidation route, whereas higher organisms utilize both the S- and the R-isomers of hydroxyacyl-CoA.

40 M. tuberculosis Rv0216 and Rv0130 The M. tuberculosis Rv0216, classified as a conserved hypothetical protein by the Pasteur Institut TubercuList server, was found to be essential for Mtb growth in vivo (Sassetti and Rubin, 2003). Based on a large-scale data mining, the 36 kDa Rv0216 was also suggested to be a unique mycobacterial protein (Cole, 2002b; Marmiesse et al., 2004), making it a potentially interesting target. The 16 kDa conserved hypothetical protein Rv0130 in turn, was selected on the basis of its remote similarity to the C-terminal half of Rv0216. Both rv0216 and rv0130 are surrounded by genes predicted to be involved in fatty acid metabolism (Cole et al., 1998). However, neither of them seems to be a part of an operon, since they are both coding in the re- verse direction compared to neighboring genes. The structures of Rv0216 and Rv0130 have been deposited at the Protein Data Bank with PDB-codes 2BI0 and 2C2I, respectively.

Structure of M. tuberculosis Rv0216 (Paper IV) Crystallization and structure determination Native and selenomethionine-substituted Rv0216 was cloned, expressed and purified by Kristina Bäckbro. Protein of both types were subsequently crystallized by Alina Castell. By comparing native and SeMet protein using mass spectroscopy, the selenomethionine incorporation was estimated to 2.6 sele- niums per 337 residues. Due to the relatively low SeMet content, efforts were made to collect a highly redundant / low intensity MAD dataset. Four datasets of a single SeMet crystal were collected at ESRF beamline ID29, at wavelengths above, below, and at the Se-absorption edge to resolutions of 2.7, 2.5, 2.8 and 1.9 Å, respectively. A native 1.9 Å dataset was later collected at ESRF beamline ID14:2. The HKL suite of programs (Otwinowski and Minor, 1997) was used to integrate and scale the images. Both SeMet and native crystals were found to belong to space group P6322, unit cell parameters a=77.9 Å, b=77.9 Å & c=178.5 Å, and a=78.8 Å, b=78.8 Å & c=180.3 Å, respectively. Assuming one monomer in the asymmetric unit 3 -1 gives a Vm of 2.2 Å Da (Matthews, 1968), corresponding to a solvent content of 44%. Two strong heavy atom peaks were identified with the Patterson-based RSPS program (Knight, 2000) using the low resolution Se-peak diffraction dataset. A third weaker site was located by difference Fourier analysis after phasing with MLPHARE (Otwinowski, 1991). Initial MAD phasing was carried out with SHARP (de la Fortelle and Bricogne, 1997) and then extended to a resolution of 1.9 Å using SAD phasing. Subsequent solvent flattening and histogram matching using DM (Cowtan and Main, 1998) im-

41 proved maps considerably. The Rv0216 model was built interactively with O (Jones, 2004) and refined against the native dataset with REFMAC5 (Murshudov, 1997) to a resolution of 1.9 Å. ARP/wARP (Perrakis, 1997) was used to locate 325 water molecules in peaks (>2ı) of the |2Fobs|-|Fcalc| electron density map. The final phasing and refinement statistics are given in Paper IV, Table 2.

Structure The Rv0216 molecule is formed by a large, highly curved ȕ-sheet packed onto four major Į-helices. Somewhat surprisingly, the structure was found to be composed of two very similar domains, an N-terminal (residues I8-S159) and a C-terminal domain (residues V199-F337) that pack side-by-side to form an elongated molecule (Figure 14). Each domain is built up by an antiparallel five stranded ȕ-sheet, wrapped around a long and a short Į-helix in a so-called single hotdog fold (SHD). This fold was originally observed in the 19 kDa Escherichia coli dehydratase-isomerase, FabA (Leesong et al., 1996). Interestingly, FabA forms a functional homodimer with a quaternary structure similar to Rv0216. The double hotdog fold (DHD) of Rv0216, formed by two SHD domains, has also been previously described; in an E. coli thioesterase II (Li et al., 2000) and two eukaryotic hydratase 2 enzymes (Koski et al., 2005; Koski et al., 2004).

Figure 14. The Rv0216 DHD monomer, formed by two very similar SHD domains.

Although the sequence similarity between the two Rv0216-domains is only about 16%, the N- and C-terminal structures are very similar and two domains are readily superimposed.

42 One of the most closely related structures to Rv0216 is an R-specific enoyl hydratase 2 from Candida tropicalis, active in the peroxisomal ȕ- oxidation of fatty acids (PDB-code 1PN2 / 1PN4 (Koski et al., 2004)). This enzyme exhibits a double hotdog fold built up by two SHD domains connected by a long linker, similar to Rv0216. The SHD dimers of a R-enoyl hydratase from Aeromonas caviae (PDB-code 1IQ6 (Hisano et al., 2003)) and a protein of unknown function from Archaeoglobus fulgidus (PDB-code 1Q6W Fedorov et al., unpublished) also exhibit a very similar quaternary structure (Paper IV, Table 3). Both the SHD and the DHD hydratases feature a large so-called overhanging lid section (Hisano et al., 2003) composed of a 3-turn helix followed by a long loop, interacting with the neighboring subunit or domain. The N- and the C-terminal domains of Rv0216 exhibit similar structures, with helices Į1 and Į5 folding onto the central hotdog helices of the C- and the N- terminal domains, respectively. The original single hotdog folded protein on the other hand, the E. coli FabA dehydratase-isomerase (PDB-code 1MKA (Leesong et al., 1996), features a very reduced lid section, consisting only of a short 1.5 turn helix and a loop.

Putative active site All known SHD dimers, including the R-specific enoyl hydratase (Hisano et al., 2003), the ȕ-hydroxyacyl dehydratase (Kimber et al., 2004), the ȕ- hydroxydecanoyl dehydratase-isomerase (Leesong et al., 1996), as well as several proteins of unknown function (Fedorov et al., unpublished) (Tajika et al., 2004)(Kim et al., unpublished), seem to feature two identical active sites located on either side of the extended ȕ-sheet. The eukaryotic double hotdog folded hydratases (Koski et al., 2005; Koski et al., 2004), in turn, exhibit only one. These potential catalytic sites are all situated at equivalent positions in a hydrophobic cavity located at the SHD-dimer or DHD-domain interfaces. By comparing a set of 49 Rv0216-like sequences (Paper IV Fig- ure 4), gathered by an iterative approach, a putative catalytic site of Rv0216 could be identified. This potential active site is formed by a set of four highly conserved polar side-chains, situated in a deep pocket coinciding with one of the catalytic sites of the SHD / DHD structures (Figure 15).

43 Figure 15. The side-chains of N91, R152, N232 and H237, conserved in all Rv0216- like sequences, form a potential Rv0216 active site cavity (dark grey), slightly similar to an A. caviae R-hydratase (light grey).

Due to the relatively high nucleotide similarity between the N- and the C- terminal domains, it seems likely that the DHD-type structure of Rv0216 arose from a duplication event of an original SHD-encoding gene. Thus, the ancestral Rv0216 might have contained two active site cavities. However, the equivalent position of such an active site, situated on the other side of the large sheet from the conserved pocket, is not retained in Rv0216 or the large set of Rv0216-like sequences. The loss of the second active site might possibly be explained by it being obscured by the long linker connecting the two domains. The active sites of the hydratases and the dehydratase / dehydratase- isomerases all feature a common catalytic histidine side-chain situated at an extended loop in the overhanging lid section (Hisano et al., 2003; Kimber et al., 2004; Koski et al., 2004; Leesong et al., 1996). In the hydratases, the active site is complemented by an aspartyl side-chain located at the same loop, five residues ahead of the histidine. The dehydratases and the dehydratase-isomerases instead use an additional glutamyl / aspartyl residue from the hotdog helix of the neighboring subunit for catalysis. A superposition of these structures onto Rv0216, results in a close fit of the conserved catalytic histidine to H237 of Rv0216. However, instead of the vicinal carboxylic acid of the hydratase / dehydratase enzymes, the putative active site pocket of Rv0216 contains a highly conserved asparagine residue, N232 (Figure 16). Although the pocket occupies about the same volume as e.g. the A. caviae R- specific hydratase, the lining of the cavity shares little or no similarity to any of the known structures. However, parts of the residues decorating the

44 pocket are highly conserved within the set of Rv0216-like proteins. The side-chains of N91 and R152 for example, are retained in all Rv0216-like sequences and might be potential candidates for involvement in substrate binding and / or catalytic activity.

Figure 16. (A) The putative Rv0216 active site (dark grey side-chains) (B) compared to the A. caviae R-hydratase active site (light grey side-chains).

Putative Rv0216 substrates? In comparison to the R-specific hydratases that act on CoA-associated acyl thioesters from fatty acid ȕ-oxidation (Hisano et al., 2003; Koski et al., 2004), the dehydratase / dehydratase-isomerases from FAS II (Takayama et al., 2005) utilize ACP-bound fatty acid derivatives in vivo (Figure 12). How- ever, both FabZ and FabA have been shown to be able to use the corresponding CoA substrate analogs in vitro (Brock et al., 1967; Sharma et al., 2003), indicating similarities between ACP and CoA binding modes. Two hotdog folded enzymes have been described in complex with a CoA- associated compound; the C. tropicalis hydratase 2 together with its product, R-3-hydroxydecanoyl-CoA (3RHDC) (Koski et al., 2004), and the Arthro- bacter sp. strain SU 4-hydroxybenzoyl-CoA thioesterase with a 4- hydroxyphenacyl-CoA moiety (4HPC) (Thoden et al., 2003). A superposition of the hydratase 2 enzyme-product complex onto Rv0216, reveals that parts of the pantetheine unit and six carbons of the C10 3RHDC acyl chain can easily be fitted into the putative substrate binding cleft (Figure 17A). The pantetheine unit, stretching up between ȕ2 and ȕ7, is potentially interacting with a number of hydrophobic side-chains, conserved in most known hydratase, dehydratase, dehydratase-isomerase and thioesterase structures. However, the following ADP-phosphate moiety, bound on the surface of the C. tropicalis enzyme, does not fit Rv0216 without conformational changes.

45 Figure 17. (A) Superposition of an R-3-hydroxydecanoyl chain into the putative active site cavity of Rv0216. (B) A conserved patch (dark grey) on the surface of Rv0216 (grey) indicates differences in binding of a potential CoA or ACP moiety compared to the hydratase 2 and the thioesterase enzymes. 3RHDC (top) and 4HPC (bottom).

Interestingly, a number of surface-residues of Rv0216, including A90, R119, K124, L132 and G131, are highly conserved within the Rv0216-like sequences (Figure 17B). This conserved patch, located on the N-terminal domain of Rv0216, indicate that an assumed CoA or ACP-unit might bind in a different way as compared to both the hydratases and the thioesterases. In- stead of being positioned in the middle of the large 10-stranded ȕ-sheet, as in the case of the C. tropicalis enzyme (Koski et al., 2004), or wedged between the two subunits as in the thioesterase case (Thoden et al., 2003), a CoA/ACP-moiety might fold perpendicular to the sheet of Rv0216, potentially interacting with the positively charged side-chains R119 & K124. Although it is clear that Rv0216 most likely does not use either 4HPC or 3RHDC as substrates, the superposition shows that Rv0216 indeed might utilize similar kind of compounds. The depth of the putative binding cavity, restricted by the conserved R152 and by the hotdog helix of the adjacent domain, indicates a 4-6 carbon substrate, whereas the relative abundance of hydrophilic residues in the binding pocket, suggests a slightly less hydrophobic substrate than a plain fatty acid chain. However, the question whether Rv0216 can bind CoA and / or ACP remains unresolved.

Rv0216 a (de)hydratase? Based on the similarities to the SHD / DHD hydratases and the SHD dehydratase / dehydratase-isomerases, the spectroscopic assay of Fukui and coworkers (Fukui et al., 1998) was used to investigate both the hydratase and the dehydratase activity of Rv0216. However, Rv0216 did not exhibit any

46 activity on either the minimal hydratase substrate, trans-2-butenoyl CoA (crotonyl-CoA), or the minimal dehydratase substrate, R-3-hydroxybutyryl- CoA. The potential catalytic mutant N232D did also not show hydratase / dehydratase activity.

Related M. tuberculosis proteins By using the 3D-PSSM threading server (Kelley et al., 2000), seven other putative single / double hotdog-domains could be identified in the translated Mtb genome (Table 1). One of these, Rv2499c, seems to be a SHD Rv0216- like protein, whereas Rv0130, Rv0636, Rv3389c, Rv3538, Rv0241 and Rv2524c, are SHD / DHD containing proteins, featuring minor variations of the so-called hydratase 2 motif [YF]-X(1,2)-[LVIG]-[STGC]-G-D-X-N-P- [LIV]-H-X(5)-[AS] (Qin et al., 2000). The only one of these proteins that has been assigned a biological function is Rv2524c, the 326 kDa fatty acid synthase I, FAS I of M. tuberculosis (Takayama et al., 2005). However, the FAS I cycle involves a dehydratase step, making a hydratase motif in Rv2524c rather interesting. This motif seems to be contained in a ~16 kDa single hotdog domain, starting around residue 1200. The preceding 150 amino acid stretch, also shows some similarity to a hotdog-related structure; the N-terminal domain of the C. tropicalis hydratase 2 enzyme (Paper IV, Figure 4). Based on these results we predicted that the dehydratase activity of Mtb FAS I might be governed by a hydratase active site, formed by a DHD-type structure. This suggestion was recently verified by the low-resolution crystal structure of both a human (Maier et al., 2006) and a fungal (Jenni et al., 2006) FAS I complex. Curiously, the enzyme performing the dehydration step in E. coli fatty acid synthase II, the classical dehydratase / dehydratase-isomerases, exhibit substantial hydratase activity. The following FAS II enzyme, enoyl-ACP reductase FabI (InhA in Mtb, Figure 12), in turn reduces the small amounts of enoyl-ACP chains produced by the dehydratases, pulling each cycle of fatty acid elongation to completion (Heath and Rock, 1995; Heath and Rock, 1996; Sharma et al., 2003). Since no FAS II ȕ-hydroxyacyl-ACP dehydratase FabZ has been identified in M. tuberculosis (Takayama et al., 2005) and only a low dehydratase activity seems to be required from such an enzyme, it is tempting to speculate that a hydratase-motif-containing protein might act also in Mtb fatty acid elongation system II. This would make any of Rv0130, Rv0636, Rv3389c, Rv3538 or Rv0241 potential candidates for being the missing mycobacterial FabZ.

47 Table 1. Rv0216-related proteins in M. tuberculosis. Rv number Pfam annotation Size (aa) Our classification active site residues Rv0216 Not present 337 DHD N232, H237 Rv0216-like Rv2499c Possible oxidase 185 SHD N66, H71 regulatory-related Rv0216-like protein Rv0130 Hypothetical 151 SHD D40, H45 protein Hydratase-like Rv0636 Hypothetical 142 SHD D36, H41 protein Hydratase-like Rv3389c Possible 290 DHD D186, H191 dehydrogenase Hydratase-like Rv3538 Probable 286 DHD D189, H194 dehydrogenase Hydratase-like Rv0241c Hypothetical 280 Probable DHD D190, H195 protein Hydratase-like Rv2524c Probable fatty 3069 Probable DHD D1229, H1234 acid synthetase Hydratase-like

48 Conclusions and future perspectives The conserved hypothetical protein Rv0216 exhibits a double hotdog fold, composed of an N- and a C-terminal single hotdog folded domain. A putative active site of Rv0216, located in a deep cavity between the two SHD domains, could be identified by comparing a number of Rv0216-like sequences from different actino- and proteobacteria. This putative catalytic site shares some similarity to three DHD / SHD R-specific enoyl hydratases and two SHD dehydratase / dehydratase-isomerases, including a conserved catalytic histidine residue. Interestingly, the proposed Rv0216 active site is not only retained in 35- 40 kDa DHD-type sequences, but also in several smaller 15-20 kDa SHD sequences. Consequently, these proteins are likely to form double hotdog- folded homodimers, with Rv0216-like active sites built up from both subunits. This observation, combined with the unique conservation pattern of Rv0216-like sequences and the data indicating Rv0216 to be an essential Mtb protein (Sassetti and Rubin, 2003), suggests that the Rv0216-like proteins are forming a distinct family, separate from both the hydratases and the dehydratases. The Rv0216-family is however not mycobacteria-specific, as indicated by Cole (Cole, 2002b) and Marmiesse (Marmiesse et al., 2004). Adding the structural similarities, as well as the placement in the M. tuberculosis genome, close to a predicted fatty acid ligase (Rv0214), an acyl-CoA dehydrogenase (Rv0215) and a putative lipid esterase (Rv0217), the Rv0216-like proteins might possibly be involved in some more general bacterial fatty acid metabolism. The finding of an SHD domain with a hydratase motif instead of a classical dehydratase motif in M. tuberculosis FAS I is interesting for reasons of broader interest. This indicates that the dehydratase activity of mycobacterial FAS II also might be governed by a hydratase active site, thus giving hints on the missing Mtb FabZ dehydratase. Several different approaches will probably be needed to elucidate the function of Rv0216. However, current studies are restricted to testing a few fatty acid related substrates and assays in collaboration with Annaik Que- mard and coworkers at the University of Toulouse.

49 Structure of M. tuberculosis Rv0130 (Paper V) Crystallization and structure determination Selenomethionine-substituted Rv0130 was cloned, expressed and purified by Kristina Bäckbro. Crystallization trials of SeMet protein were performed with JBScreen HTS (Jena Bioscience) using the sitting-drop vapour diffusion method (McPherson, 1982). Diffraction data were collected at the ESRF synchrotron, beamline ID14:3, above the Se-absorption edge to a resolution of 1.7 Å. Subsequent indexing, integration and scaling of the data were carried out using the HKL suite (Otwinowski and Minor, 1997). The crystal was found to belong to space group P21212 with unit cell parameters a=58.7 Å b=108.7 Å & c=50.4 Å, giving a Matthews coefficient of 2.5 Å3Da-1 (Matthews, 1968) when assuming two molecules in the asymmetric unit. The data was analyzed with SHELXC (Schneider and Sheldrick, 2002), indicating a high resolution cutoff of 2.0 Å for SAD phasing. Four potential selenium sites were located using SHELXD (Schneider and Sheldrick, 2002) and cross-validated using MLPHARE (Otwinowski, 1991). Initial phases were calculated by SHARP (de la Fortelle and Bricogne, 1997) and subjected to density modification in DM (Cowtan and Main, 1998), but were not good enough for main-chain tracing. Since the positions of the 2+2 Se-atoms were not sufficient to calculate the NCS operators relating the two molecules, a fifth dummy atom was roughly placed on the assumed NCS twofold axis using several SHD / DHD models and O (Jones, 2004). The resulting ap- proximate operator was then refined using a full 6-dimensional search in Imp (Jones, 1992). Subsequent two-fold averaging in DM improved maps considerably. ARP/wARP (Perrakis, 1997) was used for automatic chain tracing. Crystallographic refinement was done in REFMAC5 (Murshudov, 1997) to a resolution of 1.8 Å. After each cycle of refinement, the ıA- weighted |2Fobs|-|Fcalc| and |Fobs|-|Fcalc| maps (Read, 1986) were used for further model rebuilding in O. Final refinement statistics are given in Paper V, Ta- ble 1.

Structure As predicted, Rv0130 exhibits a single hotdog fold. The Rv0130 monomer structure is composed of a five-stranded anti-parallel ȕ-sheet packed on top of a long and a short Į-helix, flanked by a small two-stranded parallel ȕ- sheet. As in Rv0216, a number of ȕ-bulges curve the large sheet to fit the long hotdog helix. The small parallel sheet in turn, situated at the C-terminal edge of the central helix, does not have any counterparts in any of the known SHD / DHD structures. Two Rv0130 molecules pack side-by-side, forming a double hotdog folded dimer with an extended 10-stranded sheet (Figure 18A). As in the R-hydratases and Rv0216, helix Į1 and the long following

50 loop, crosses over the hotdog helix to form a lid structure that interacts with the second subunit. This conformation creates a four-helix bundle on the concave side of the extended sheet, with two helices from each of the two dimer molecules. The four-helix bundle stabilizes the dimer by a large number of hydrophobic interactions, burying a total solvent accessible area of more than 2000 Å2 (PDBsum, (Laskowski et al., 2005)).

Figure 18. (A) The Rv0130 homodimer (B) In Rv0130, the hotdog helix is distorted by a central proline residue as well as a number of sequence changes, leading to a distinct 25° kink of the helix axis.

In both the SHD and DHD proteins, the hotdog helices pack at about 45º on the large ȕ-sheet. However, in Rv0130, the axis of the corresponding central helix is broken. This sharp kink is caused by a number of changes in the packing of the helix onto the ȕ-sheet as well as a proline residue (P71), situated in the middle of the long helix (Figure 18B). The broken helix is followed by a 10-residue stretch that folds onto the N-terminus, forming the small two-stranded parallel ȕ-sheet. Even though none of the known hotdog structures exhibit similar changes or a similar proline insertion, the N- terminal domain of the eukaryotic DHD hydratase 2 structures 1PN2 and 1S9C feature a slightly similar distortion; a hotdog helix corrupted by a short loop region (Figure 19). The distortion of the hotdog helix opens up the domain interface, creating a long tunnel harboring the catalytic residues of these enzymes (Koski et al., 2005; Koski et al., 2004). In Rv0130 the break- ing of the helix causes a similar effect, a curved tunnel formed by the dimer interface (Figure 20). The lower part of the tunnel, restricted by the C- terminal portion of the hotdog helix and the small ȕ-sheet, occupies about the same volume as in the DHD enzymes. However, in Rv0130 the end of the tunnel is deeper and slightly more polar than its eukaryotic counterparts. The rest of the tunnel, on the other hand, created by the lid section and strand ȕ2, is more similar to the substrate binding pocket of the A. caviae hydratase

51 and the putative substrate binding cavity of Rv0216, lined by conserved residues like H45, G63 and P99.

Figure 19. (A) The hotdog helices of the A. caviae SHD R-hydratase, (B) Rv0130 and (C) the C. tropicalis DHD hydratase 2.

Figure 20. The broken hotdog helix of Rv0130 opens up the active site cavity to create a 20 Å open ended tunnel.

Catalytic activity of Rv0130 The large overhanging lid section of Rv0130 is very similar to the SHD R- specific enoyl hydratase as well as the C-terminal domains of the eukaryotic DHD hydratase 2 structures and Rv0216. As predicted from sequence alignments, Rv0130 contains a well-conserved hydratase motif, situated in the middle of the long tunnel formed by the overhang and the neighboring subunit. A superposition of Rv0130 and the A. caviae R-hydratase shows a close fit of Rv0130 H45 and D40 to the active His-Asp pair of the hydratase, as well as several of the residues lining the pocket (Figure 21).

52 Although Rv0130 is only remotely related to the dehydratase / dehydratase-isomerases, the putative active site also shares some similarity with the P. aeruginosa FabZ (Kimber et al., 2004), as well as the E. coli FabA (Leesong et al., 1996). A superposition of the catalytic histidines of the FabA / FabZ enzymes onto H45 of Rv0130, reveals that the catalytic carboxylate groups of the FabA / FabZ structures and D40 of Rv0130 are structurally conserved. Based on this remote similarity, the similarity to the R-hydratases and the apparent ambiguity of the hydratase motif discussed previously, both the hydratase and the dehydratase activity of Rv0130 were investigated. Rv0130 was found to be able to catalyze the hydration of trans-2-butenoyl- 2 -1 CoA to 3-hydroxybutyryl-CoA with a kcat of 1.1 x 10 s and a Km of 55 ȝM. The stereo-specificity for the hydration reaction was not investigated. However, the overall similarity of the Rv0130 active site to both the SHD and DHD R-hydratases compared to the inverted active sites of classical S- hydratases (crotonases) (Engel et al., 1996), suggests Rv0130 to be an R- specific enzyme. Site-directed mutagenesis subsequently confirmed D40 to -1 be important for hydratase activity, reducing kcat to 0.057 s . The H45Q mutant in turn, was completely inactive. Rv0130 showed only low dehydratase activity using R-3-hydroxybutyryl-CoA as a substrate, and kinetic val- ues could not be determined.

Figure 21. (A) The Rv0130 active site compared to (B) the A. caviae R-hydratase.

Pockets, prolines and substrate specificity The tunnels of both Rv0130 and the eukaryotic DHDs are formed as a result of a distorted hotdog-helix followed by a long connection to the central ȕ- strand (Figure 19). In the two eukaryotic DHD hydratases, this tunnel en- ables the enzymes to take part in the ȕ-oxidation of long (Koski et al., 2004) and branched (Koski et al., 2005) fatty acid chains. It is quite likely that

53 Rv0130 also is able to use longer CoA-thioester chains than the crotonyl- CoA tested. The substrate specificities of four different R-hydratases from P. aeruginosa, PhaJ1-PhaJ4, were recently described by Tsuge and coworkers (Tsuge et al., 2003). Three of these enzymes, PhaJ2, PhaJ3 and PhaJ4 were able to hydrate C8-C12 enoyl-CoA chains, while PhaJ1 was found to be active only on C4-C6 enoyl-CoAs. A sequence alignment shows that PhaJ2 and PhaJ3 are likely to feature DHD-type structures (Paper V, Figure 3). Both putative domains of these enzymes are similar to the eukaryotic hydratase 2 enzymes, featuring a long connection between the N-terminal hotdog helix and strand ȕ2. Compared to the pocket-containing DHD Rv0216, PhaJ2 and PhaJ3 have 10 and 13 extra residues between the beginning of the hotdog helix and the conserved ȕ-bulge on the central strand. This might be indicative of a substrate binding tunnel instead of a pocket, thus explaining the apparent long-chain substrate specificity. PhaJ4 on the other hand has only five extra residues compared to Rv0216 in the same region. However, this enzyme seems to feature a proline residue, P70, positioned in the middle of the hotdog helix, possibly creating a substrate binding tunnel similar to Rv0130. The only short-chain enoyl hydratase PhaJ1 of the study seems to exhibit an SHD fold with a minimal 21-residue hotdog helix (Paper V, Table 3), which correlates well with a closed substrate binding crevice. Of the six other M. tuberculosis proteins predicted to exhibit a hotdog- type structure related to Rv0216 (Table 1), Rv3389c, Rv3538, Rv2524c and Rv0241 are more similar to the eukaryotic hydratase 2 enzymes than to both Rv0130 and the A. caviae 1IQ6. These proteins all seem to have an extended loop following the N-terminal hotdog helix, suggesting a substrate binding tunnel. The predicted SHD homodimers, Rv0636 and Rv2499c, on the other hand, are more closely related to 1IQ6. Both these structures seem to feature a short loop between the hotdog helix and the central ȕ-strand (Paper V, Table 3).

PHA biosynthesis The four R-specific enoyl hydratases investigated by Tsuge and coworkers (Tsuge et al., 2003) share between 11% (PhaJ3) and 43% (PhaJ4) sequence identity with Rv0130. These proteins, as well as the A. caviae 1IQ6 enzyme, are all involved in polyhydroxyalkanoate (PHA) biosynthesis. Polyhydroxy- alkanoates are biopolymers composed of hydroxy fatty acids polymerized by PHA synthase, PhaC. These PHA polyesters are accumulated in intracellular granules by a wide range of gram-positive and gram-negative bacteria for storage of carbon and reducing power (Madison and Huisman, 1999). It is not known whether M. tuberculosis is able to produce PHAs. However, M. tuberculosis Rv1683 shows e.g. 27% sequence identity (E-value 1e-20) to the PHA synthase PhaC of Chlorogloeopsis fritschii (Hai et al., 2001) over a 340 amino acid stretch, including 9 of 15 residues that Madison and Huis-

54 man (Madison and Huisman, 1999) reported to be absolutely conserved in 26 experimentally characterized PHA polymerases. In addition, Rv1683 seems to exhibit an extra domain, sharing similarity to a very long-chain acyl-CoA synthetase from H. sapiens (Steinberg et al., 1999) (25% identity over a 440 aa stretch, E-value 7e-29). Interestingly, Rv1683 is present in M. leprae (ML1346) and was found to be required for mycobacterial growth in vitro (Sassetti et al., 2003).

55 Conclusions and future perspectives As suspected from threading results, Rv0130 features a single hotdog fold, forming a double hotdog folded dimer. In contrast to Rv0216, Rv0130 contains a well-conserved hydratase active site and also exhibit substantial hydratase activity. The Rv0130 active site is situated in a curved tunnel formed by a sharp kink in the Rv0130 hotdog helix. Four of the six previously predicted SHD / DHD containing M. tuberculosis proteins (Table 1), may feature a similar long substrate-binding tunnel. It is likely that Rv0130, as well as several of these proteins, are able to act on long fatty acid chain derivatives. Rv0130 could theoretically be involved in either fatty acid biosynthesis by acting as a FabZ dehydratase, or for example in a new R-specific branch of the fatty acid ȕ-oxidation by converting trans-enoyl-CoA to R- hydroxyacyl-CoA (Figure 13). However, since Rv0130 shows such a high hydratase over dehydratase ratio in vitro as well as a high similarity to the PhaJ enzymes, it is tempting to speculate that Rv0130 instead is involved in e.g. PHA production, supplying hydroxyacyl-CoA monomers to an Mtb PhaC enzyme. Such a hypothesis could possibly be substantiated by investi- gating the ability of Rv0130 and Rv1683 to synthesize PHAs from ȕ- oxidation precursors in PHA-negative mutants of E. coli as has been done for some of the PhaJ enzymes (Park and Lee, 2003). To investigate the substrate specificity of Rv0130, enzymatic studies using long-chain CoA-thioesters are currently underway in collaboration with Annaik Quemard, University of Toulouse. However, it cannot be ruled out that Rv0130 also acts on ACP-associated thioesters, since CoA and ACP binding sites often overlap.

56 Summary in Swedish

Denna avhandling sammanfattar studierna kring fyra olika proteiner från två olika organismer. Den första delen berör ett så kallat xyloglucan endotransglycosylas, ett enzym med förmågan att klippa och klistra ihop långa socker- molekyler i växters cellväggar. Den andra delen handlar om det tredimensionella utseendet av tre proteiner med okänd funktion från tuberkulosbakterien Mycobacterium tuberculosis (Mtb). Genom att skjuta oerhört koncentrerad strålning på en liten kristall av ett protein och sedan registrera hur strålarna bryts, kan en tredimensionell struktur beräknas med mycket god nogrannhet. Denna metod, s.k. röntgenkristal- lografi, har använts för att bestämma strukturen av de fyra olika proteinerna. Eftersom ett proteins struktur är nära förknippat dess funktion, kan sådana studier ge en detaljerad inblick i hur proteiner verkar och interagerar med specifika kemiska föreningar, s.k. substrat. Växters cellväggar är uppbyggda av en komplicerad matris av cellulosa, lignin och proteiner. Olika sockerpolymerer, till exempel xyloglucan, kan i sin tur binda till cellulosa och skapar därigenom ett stadgande nätverk mel- lan närliggande cellulosafibriller. När celler växer måste dock detta nätverk byggas om. Ett flertal proteiner är inblandade i denna ommodelleringspro- cess, bl.a. ett enzym, xyloglucan endotransglycosylas (XET), som inte bara kan hydrolysera (klippa) xyloglucanlänkar, utan också ligera ihop en av än- darna med en ny sockermolekyl, en så kallad acceptor. Den tredimensionella strukturen av ett XET-enzym från Darrasp veckas till ett s.k. ’ȕ-jelly roll fold’, liknande ett antal enzymer med enbart hydroly- tisk aktivitet. XETs substratbindningssite skiljer sig dock signifikant från de hydrolytiska enzymerna och ger en möjlig förklaring till varför enzymet inte bara utför hydrolys, utan också ligering av xyloglukanmolekyler; När sock- ermolekylen klipps och den halva xyloglukankedjan lämnar XET inaktiveras enzymet. Detta leder till att den andra delen av sockerkedjan hålls kvar tills en acceptormolekyl binder på den tomma platsen. Detta återaktiverar i sin tur enzymet och ligering kan utföras. Tuberkulosbakterien M. tuberculosis orsakar årligen över en och en halv miljon dödsfall och är åter på frammarch, trots modernare sjukvård och för- bättrade levnadsvillkor. En av orsakarna är HIV/AIDS snabba utbredning i framförallt Afrika och Östeuropa. En annan är utvecklingen av antibiotikare- sistens, vilket avsevärt försvårar och förlänger eventuell behandling. För

57 närvarande bär nästan en tredjedel av världens befolkning på tuberkulosbakterien och åtta miljoner människor insjuknar varje år i TBC. För att bättre förstå tuberkulosbakteriens livscykel och för att underlätta utvecklingen av nya läkemedel mot TBC, har bakteriens arvsanlag bestående av cirka 4000 gener nyligen sekvenserats. Bara ungefär hälften av dessa geners funktion har dock kunnat identifieras. Av de gener vars funktion har kunnat förutsägas, verkar en oproportionerligt stor andel vara inblandade i uppbyggnaden och nedbrytningen av olika typer av fettsyror. Dessa verkar bland annat vara viktiga för bakteriens cellvägg som är uppbyggd av en rad ovanliga fetter, sockerarter och proteiner, vilket i sin tur gör Mtb ovanligt motståndskraftig mot angrepp utifrån. En annan proteinfamilj som verkar vara kraftigt överrepresenterad hos tuberkulosbakterien är s.k. epoxidhydrolaser (EHs). Epoxidhydrolaser är livs- nödvändiga för många organismer då de bryter ned skadliga restprodukter, epoxider, som bildas i cellerna. Vissa bakterier använder inte bara epoxidhydrolaser till detoxifiering, utan också till energiproduktion då resurserna är knappa. Jämfört med till exempel E. coli har M. tuberculosis nära fem gång- er så många epoxidhydrolaser, vilket indikerar att de har en viktig funktion i bakteriens livscykel. Tillsammans med en forskargrupp i Schweiz har vi identifierat och bestämt den tredimensionella strukturen hos ett av dessa enzymer, Rv2740, tidigare klassificerat som ett okänt protein. Rv2740 veckas till en konformad molekyl bestående av ett stort krökt betaflak (Figur 10A). Betaflaket skapar i sin tur ett aktivt centrum på botten av en djup ficka, vilket liknar det aktiva centret av en annan redan känd epoxidhydrolas, LEH (Figur 11AB). Storleken och utseendet på de två fickorna indikerar dock att Rv2740 potentiellt kan bryta ned mycket större substrat än LEH, till exempel olika typer av fettsyra- och sterolderivat. Fortsatta studier är inrik- tade på att försöka bestämma Rv2740s naturliga substrat och därigenom dess funktion i tuberkulosbakterien. Två andra tidigare okända proteiner från M. tuberculosis har också stude- rats, Rv0216 och Rv0130. Den tredimensionella strukturen av Rv0216 veckas till ett sk. ’double hotdog fold’ (DHD) (Figur 13) medan Rv0130 veckas till ett liknande, men hälften så stort, ’single hotdog fold’ (SHD). Vid en jämförelse av Rv0216 med andra tidigare kända strukturer, hittades några liknande strukturer med samma fold men inga nära släktingar. Däremot hittades ett stort antal mycket liknande proteinsekvenser från en rad olika organismer, alla av okänd funktion. Detta tyder på att Rv0216 är det första ex- emplet på en helt ny proteinfamilj. Rv0130 å andra sidan visade sig vara nära släkt med en familj av enzymer inblandade i hydrering av omättade fettsyror. För att ytterligare karaktärisera funktion och substratspecificitet för Rv0130 och få idéer till en funktion till Rv0216, har vi nyligen startat ett sammarbete med en forskningsgrupp i Frankrike (Universitetet i Toulouse) som specialicerat sig på M. tuberkulosis fettsyrametabolism.

58 Acknowledgements

These results would not have been possible without the help, the support, the knowledge, the patience, the whining, the generosity, the encouragement and the friendship of a large number of amazing people;

Alwyn, for (literally) dragging me to the other side of the forest and intro- ducing me to this world-O-wonders, for support and for having a song for every occasion. Sherry, for all help, support and genuine scientific enthusiasm. Gerard (yes, really tall guy) for help and great Tällberg weather. Tex, for crystallization tricks and keeping law and order in D11 (aka Dodge-city). Torsten for endless enthusiasm (and promises of a new life with less..). Seved, for 'uppställning!'. Jerry for dancing and color-coordinated shoes. Mark for advice on hardware (& mistaking me for a computer guy). Erling and Christer for invaluable support. Mats & Gunnar for help and GH in- put. Nina for magic green protein fingers & kiwi-breaks. Many thanks to all collaborators; Harry, Hongbin & Tuula at KTH, Åsa for Proctor Academy, Erni and Annette (now in Zürich).

Thanks to all past and present inhabitants of the BMC concrete castle; Emma (truly wonderful person) for everything from Max-trips & graphol- ogy-grading to late Knutby-discussions. Dennis for organized code. Jimmy for sport and good company. Henke for skating & HMM practicals. Ulrika for politically incorrect dreams. EvaLena (cool girl) for healthy skepticism. Talal for style. Martin (Dr M) for revolutionary and non rävolusjånäry discussions. The girls; Annette, for amazing agility (and sounds to go with it..). Alina (Mr Rastawoman) for girl power & scary dreams. Lena for late esrf sessions and corridor rugby. Christofer (and his real friends) for sushi & DnB. Linus for setting a cake-standard. Daniel for helping ye’ all against “dry tongue, wet footgear, foul mood and halitosis”. Henrik for optimism. Wojciech for red wine. Martin (aka Pieter) for Max-trips and 500kr bills. Magnus for Danzig & snowman-euthanasia. Jonas for being politically correct (!).

The rest of the people at ICM, SLU and biophysics; Sara (pop-culture guru) for listening, TP & dancing on the roof in Tällberg. Janos for new scientific and unscientific perspectives. Anke for parapsychologic crystallography. Remco, Nic, David, Calle, Magnus, Alexandra, Michiel, Karin, Lars,

59 Anna, Adrian, Pavel, Ingrid, Hasse, Ulla, Inger, Margareta, Stefan, Martin, Tom, Malin, Urszula, Martin, Nisse, Linda, Anton, Saeid, Agata, Andrea, Lotta, Sanji, Anatoly.. Louise for keeping me company in the rain. Wimal for always smiling. Anna J for lists & stealing my stolen mug. Fredrik for always looking busy. Kaspars for (safe) driving. Marian for skating. The Åqvist group, Johan, Stefan, Hugo, Martin, Martin, Mar- tin.. Jens for being a good judge of character. Sinsia for the too-many-days- above-5000m-face (+the half-day gringo-breakfast at Jack's afterwards).

The outdoor gang; Al, for beer, climbing, logistics, skating, insane ideas, driving lessons (did I mention beer?). Jenny for stripes, rastastyle & skating promises (2007; 4.59..!). Gösta (Norrköping mountainman) for clever climbing solutions and never-ending Yukiko-enthusiasm. Nisse for putting his foot down, climbing & keeping Annette from BMC. Fariborz for discussions about everything between ice axes & hadithic teachings. Shawn, Kristin, Tina, Emil, Kalle, Kalle, Patrick, Joel, Hedvig, Tobias, Micke for Stallet. Magnus & Magnus for new applications for risgrynsgrötkorvar.

The extended Djäknegatan crowd; Johanna for möbelberg & housing solutions, Niklas for caucasians and grillfrukost, Sara & Björn for plant dona- tions, Nina for birthday plans and Pär & Jenny for blåbärspannkakor. Daniel (Hr Bergenstråle) for bravery & late sessions. All Måns-people; Benny, Anneli, Anna-Karin, Annika, Mia.. ‘take me home country roads’.

My family, Mamma & Pappa for constant support. Nicklas for julrim-slam and putting things into perspective. Jörgen for ‘glasögonbågens böjning..’, reindeer-charming & 32-piece sushi-draw.

Åsa (*) for patience, äppelmust and subtle weather metaphors.

60 References

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