Structural Insights Into Decreased Enzymatic Activity Induced by an Insert Sequence in Mannonate Dehydratase from Gram Negative Bacterium

Journal of Structural Biology 180 (2012) 327–334

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Journal of Structural Biology

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Structural insights into decreased enzymatic activity induced by an insert sequence in mannonate dehydratase from Gram negative bacterium

Xiaoting Qiu, Yuyong Tao, Yuwei Zhu, Ye Yuan, Yujie Zhang, Hejun Liu, Yongxiang Gao, ⇑ ⇑ Maikun Teng , Liwen Niu

Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, PR China article info abstract

Article history: Mannonate dehydratase (ManD; EC4.2.1.8) catalyzes the dehydration of D-mannonate to 2-keto-3-deoxyg- Received 30 April 2012 luconate. It is the third enzyme in the pathway for dissimilation of D-glucuronate to 2-keto-3-deoxygluc- Received in revised form 21 June 2012 onate involving in the Entner–Doudoroff pathway in certain bacterial and archaeal species. ManD from Accepted 26 June 2012 Gram negative bacteria has an insert sequence as compared to those from Gram positives revealed by Available online 14 July 2012 sequence analysis. To evaluate the impact of this insert sequence on the catalytic efﬁciency, we solved the crystal structures of ManD from Escherichia coli strain K12 and its complex with D-mannonate, which Keywords: reveal that this insert sequence forms two a helices locating above the active site. The two insert a helices Mannonate dehydratase introduce a loop that forms a cap covering the substrate binding pocket, which restricts the tunnels of sub- Gram negative bacteria Insert sequence strate entering and product releasing from the active site. Site-directed mutations and enzymatic activity Substrate entering tunnel assays conﬁrm that the catalytic rate is decreased by this loop. These features are conserved among Gram Glucuronate metabolism negative bacteria. Thus, the insert sequence of ManD from Gram negative bacteria acts as a common indu- Entner–Doudoroff pathway cer to decrease the catalytic rate and consequently the glucuronate metabolic rate as compared to those from Gram positives. Moreover, residues essential for substrate to enter the active site were characterized via structural analysis and enzymatic activity assays. Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.

1. Introduction species (Mekjian et al., 1999; Rakus et al., 2007; Robert-Baudouy et al., 1982; Shulami et al., 1999). ManD is the third enzyme in D-glucuronate is a six-carbon acidic sugar that is abundantly the pathway for dissimilation of D-glucuronate to 2-keto-3-deoxyg- distributed in carbohydrate chain of proteoglycan in many animal luconate (2-KDG) involving in the Entner–Doudoroff pathway and plant tissues (Kuehl and Murphy, 2003; Murarka et al., 2010), (Murarka et al., 2010). In this step, ManD catalyzes the removal of where it serves as the sole carbon source of several endogenous one water molecule from C2-hydroxyl and C3-hydroxyl of bacterial species (Lawford and Rousseau, 1997). All macromolecu- D-mannonate to yield 2-KDG (Rakus et al., 2007; Zhang et al., lar components required by these bacteria are synthesized from 2009), which is then cleaved to pyruvic acid and glyceraldehyde D-glucuronate. Therefore, glucuronate metabolism is critical for 3-phosphate (Murarka et al., 2010). These compounds conse- the growth of these bacteria. Catabolism of D-glucuronate occurs quently participate in the tricarboxylic acid cycle and gluconeogen- via the Entner–Doudoroff pathway. Expressions of enzymes partic- esis to yield energy and precursors for polysaccharide synthesis, ipating in this pathway are induced by glucuronate and repressed respectively (Nelson and Cox, 2000). When living in its natural hab- by glucose (Mekjian et al., 1999), which suggests that the Entner– itat (the large intestine),Escherichia coli, as a model species for Gram Doudoroff pathway uses D-glucuronate as the substrate only if bac- negative bacteria, can utilize glucuronate as the sole carbon source teria live in an environment that contains glucuronate as the sole for growth. The Entner–Doudoroff pathway using D-glucuronate as carbohydrate component. the substrate is therefore critical for the colonization of E. coli in the The gene uxuA encodes mannonate dehydratase (ManD; large intestine (Peekhaus and Conway, 1998). EC4.2.1.8), which has been found in certain bacterial and archaeal Bacterial ManDs are structurally assigned to the xylose isomer-

ase-like superfamily, member of which contains a modiﬁed (ba)8 Abbreviations: ManD, mannonate dehydratase; 2KDG, 2-keto-3-deoxygluconate. ⇑ Corresponding authors. Address: 96 Jinzhai Rd., School of Life Sciences, TIM barrel (Zhang et al., 2009). Sequence alignment demonstrates University of Science and Technology of China, Hefei, Anhui, PR China. that ManD from Gram negative bacteria has an insert sequence as E-mail addresses: [email protected] (M. Teng), [email protected] (L. Niu). compared to Gram positive ones. The conservation of this insert

1047-8477/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsb.2012.06.013 328 X. Qiu et al. / Journal of Structural Biology 180 (2012) 327–334 sequence in ManD from Gram negative bacteria suggests that it has polyethylene glycol 3350 at 287 K. Before data collection, the crys- a functional importance. Structures of ManDs from two Gram po- tal was quick-soaked in a cryo-protectant solution containing 25% sitive bacteria have been solved (PDB codes 3FVM and 1TZ9), but (v/v) glycerol, 0.1 M MgCl26H2O, 0.1 M Tris–HCl, pH 8.5, 25% (w/v) there is no structural information on ManD from Gram negative polyethylene glycol 3350 and flash-cooled in liquid nitrogen at bacteria reported. To elucidate the impact of this insert sequence 100 K. X-ray diffraction data were collected on beamline 17U1 at of ManD from Gram negative bacteria on the catalytic activity, the Shanghai Synchrotron Radiation Facility (SSRF) using a MX- crystal structures of ManD from E. coli strain K12 (EcManD) and 225 CCD detector (MAR Research). The crystal-to-detector distance its complex with D-mannonate were determined. was kept at 270 mm and the crystal was rotated through a total of The structure reveals that two additional helices are formed by 120° with 0.2° rotation per frame over 1.2 s. The crystal diffracted the insert sequence of EcManD as compared to those from Gram to a resolution of 2.30 Å. The data were indexed, integrated and positives. As a consequence, the loop following the insert helices scaled with HKL2000 package (Otwinowski and Minor, 1997). adopts a different trajectory, which covers the enzymatic active Space group was determined as P21212 via analysis of the system- site. By mutagenesis, we confirmed that the loop restricts the tun- atic absences in the diffraction data. For collection of enzyme–sub- nels of substrate entering and product releasing from the active strate complex data, a crystal of EcManD was soaked in a solution site to achieve a low catalytic rate, which may shed light into containing 0.2 M D-mannonate, 0.1 M Tris–HCl, pH 8.5, 25% (w/v) understanding of the glucuronate metabolism related biological polyethylene glycol 3350 at 287 K for 20 sec, followed by quick- process in Gram negative bacteria. soaking in the cryo-protectant solution described above. X-ray diffraction data were collected on beamline 17U1 at the SSRF using an ADSC Quantum 315r detector (Area Detector System Corporation). 2. Materials and methods The crystal-to-detector distance was kept at 300 mm and the crystal was rotated through a total of 130° with 0.5° rotation per frame 2.1. Cloning, expression and purification over 1.0 s. The crystal diffracted to a resolution of 2.35 Å. The data were indexed, integrated with iMOSFLM (Leslie, 2006) and scaled The open reading frame of full length EcManD (GenBank acces- with Scala (Evans, 2006) from the CCP4 suite (Collaborative sion number: BAA02590.1), which was obtained via PCR from the Computational Project 4, 1994). The final statistics of data collec- E. coli strain K12 genome was cloned into pET28a (Novagen) ex- tion and processing are tabulated in Table 1. cised using NdeI and XhoI to create recombinant EcManD with an N-terminal hexahistidine tag (MGSSHHHHHHLVPRGSH). A single colony of BL21 (DE3) bacteria harboring the expression vector 2.3. Structure determination and refinement was cultured in 8 mL Luria–Bertani broth overnight, and then used to inoculate 0.8 L media containing 50 lg/mL kanamycin. Cells The highest probability of four monomers per asymmetric unit 3 1 were grown at 310 K for 2.5 h until OD600nm reached 0.5, and then suggested a Matthews coefficient of 2.23 Å Da (Matthews, protein expression was induced for 20 h with 0.25 mM isopropyl- b-D-thiogalactoside (IPTG) at 289 K. The bacteria were harvested Table 1 and resuspended in 40 mL binding buffer (20 mM Tris–HCl, pH Data collection and refinement statistics. 8.0, 500 mM NaCl). After disrupting the cells by sonication, bacteria were centrifuged at 15200g for 0.5 h. The supernatant recovered EcManD EcManD–D- mannonate clean lysate was loaded onto Ni-NTA agarose resin (GE Healthcare), which was pre-equilibrated with binding buffer. The tagged pro- Data collection Wavelength (Å)a 0.9792 0.9792 tein was eluted with 30 mL binding buffer including 500 mM imid- Space group P21212 P21212 azole, which was then concentrated for further purification using a Unit-cell parameters (Å, °) a = 158.96, a = 159.47, Superdex 75 gel filtration chromatography column (GE Health- b = 238.46, c = 54.35 b = 238.58, c = 54.47 care), eluted with binding buffer. The retention volume corre- Resolution range (Å) 50.00–2.30 50.00–2.35 sponding to the target protein indicates that it is monomer in Total No. of observations 444893 420228 No. of unique reflections 92683 86788 solution (Fig. S1A). The fractions corresponding to the peak were Completeness (%) 99.7 (95.7)a 99.1 (99.9) pooled and exchanged with low salt buffer (20 mM Tris–HCl, pH Average I/r (I) 27.6 (6.5) 7.4 (2.3) b 8.0, 100 mM NaCl), then further purified using a Q-sepharose Fast Rmerge (%) 0.060 (0.176) 0.167 (0.775) Flow chromatography column (GE Healthcare), eluted with a linear Refinement Resolution range (Å) 48.42–2.30 49.55–2.35 gradient of NaCl from 0.1 to 1.0 M. Fractions corresponding to the c d Rwork (%)/Rfree (%) 18.3/21.9 18.9/23.6 target protein were pooled and concentrated to 82 mg/ml. Expres- Number of non hydrogen 12647 12647 sion vectors of mutant proteins were constructed utilizing a Quik- protein atoms Change site-directed mutagenesis kit (Stratagene). Mutant Number of waters 889 878 2 proteins were expressed and purified as described above for Average B factor (Å ) 14.7 16.2 RMS bonde (Å) 0.009 0.010 wild-type. Examination of the purified protein by SDS–PAGE re- RMS anglee (°) 1.144 1.238 vealed a single band corresponding to the expected molecular Ramachandran analysisf weight of 47 kDa. Protein concentration was measured with the Favored (%) 96.6 96.2 BCA Protein Assay Kit (Pierce). Allowed (%) 3.0 3.4 Outlier (%) 0.4 0.4

a Values in parentheses are for the highest resolution shell (2.34–2.30) for native 2.2. Crystallization and X-ray diffraction data collection and processing data and (2.48–2.35) for complex data. b Rmerge = RhklRi |Ii(hkl) hI(hkl)i|/RhklRi Ii(hkl), where Ii(hkl) is the observed Preliminary screening for initial crystallization conditions was intensity of a reflection and hI(hkl)i is the mean intensity of reflection hkl. c performed by the sitting-drop vapour-diffusion method using In- Rwork = R|Fo| |Fc|/R|Fo| where Fo and Fc are the observed and calculated structure factors. dex-1, -2 (Hampton Research) at 287 K by mixing 1 ll of 82 mg/ d Rfree is calculated for a set of randomly chosen 5% of reflections prior to ml protein solution with an equal volume of reservoir solution in refinement. 48-well plates. Plate crystals were obtained from the condition e Root-mean square-deviation (RMSD) from ideal values. f containing 0.1 M MgCl26H2O, 0.1 M Tris–HCl, pH 8.5, 25% (w/v) Categories were defined by MolProbity. X. Qiu et al. / Journal of Structural Biology 180 (2012) 327–334 329

1968) and an estimated solvent content of 44.8%. The initial phases in a14 helix (termed according to EcManD) construct an extensive of structure factors were obtained by molecular replacement using interface by forming several hydrogen bonds, pi–pi conjugation the crystal structure of ManD from Streptococcus suis serotype 2 and hydrophobic interactions, which justifies the dimerization of (SsManD, PDB code 3FVM; 37% sequence identity) as a search SsManD and EfManD in solution. However, in EcManD, the resi- model via Phaser (McCoy et al., 2007). The electron density was dues at the corresponding region changes dramatically and those clear for the majority of the molecules, allowing rounds of manual dimerization interactions disappear. For example, the hydrogen building and automatic refinement using COOT (Emsley and Cow- bonds between Tyr336 and Glu3420(the adjacent monomer), tan, 2004) and REFMAC5 (Murshudov et al., 1997), respectively. Thr341 and Leu3320, Thr335 and Thr3350 in SsManD do not exist TLS refinement was executed in REFMAC5 at the final stage of in EcManD (Fig. S1B, C). Also, the pi–pi conjugation between aro- refinement. For determination of the EcManD–D-mannonate com- matic rings of Tyr328 and Tyr3280 in SsManD is eliminated in Ec- plex structure, the EcManD apo-form model was refined against ManD by the substitution of Tyr328 with Ile371. In addition, the data of enzyme–substrate complex by REFMAC5. After several Pro23 and Met338 locating in the dimeric hydrophobic interface cycles of refinment, continuous electron density in FO–FC map of SsManD are changed into Gly23 and Lys375 in EcManD, respec- was found at the active sites of the two EcManD monemers in tively. In summary, substitutions of residues in a14 helix result in the asymmetric unit, which was assigned as D-mannonate indi- EcManD being a monomer in solution. As substitutions of those cated by its well fit with electron density. While the other two residues contributing to dimerization in Gram positive ManDs monomers in the asymmetric unit present weak and discontinuous are conserved among Gram negative ones (Fig. S3), the oligomeri- electron density at the active sites and thus substrates of these two zation states of other Gram negative ManDs are probably the same monomers are excluded in the final model. The probable reason for as that of EcManD. the different D-mannoate occupancies in the four monomers per asymmetric unit is crystal packing (Fig. S2). In the final models 3.2. Metal ions in the active site of EcManD and EcManD–D-mannonate complex, all the EcManD residues (1–394) are visible except the last C-terminal residue in ManD has been reported to require a metal ion to recognize and three of the four monomers per asymmetric unit. The stereochem- orientate the substrate within the active site and stabilize the eno- ical quality of the final model was checked by MolProbity (Lovell late intermediate formed during the dehydration reaction (Zhang et al., 2003). The final results of model building and refinement et al., 2009). Moreover, the activity of ManD appears to be metal are listed in Table 1. All structure figures were prepared with Py- ion type dependent with the Mn2+ being the optimum one (Dreyer, MOL (DeLano, 2008). 1987). During the structure refinement, a positive spherical elec-

tron density was found in the active site of EcManD in FO–FC 2.4. Metal ion analysis map. The distances from the center of this spherical density to sur- rounding atoms (Ne/His233, Sc/Cys271, Nd/His298, Od/Asp 351 Purified EcManD diluted to 10 mg/ml were treated with 75% (v/ and a water oxygen atom) range from 2.0 to 2.5 Å, which are within the limit of coordinate bond length. These residues are identical v) HNO3 for metal element determination by inductively coupled 2+ plasma atomic emission spectroscopy (ICP-AES). An equal volume with the coordinating residues of Mn in SsManD. Thus, this pos- 2+ of buffer was used as a control. The assay was carried out at the iton is deduced to be occupied by a Mn ion, which is verified by Physical and Chemical Scientific Experimental Center of University ICP-AES. of Science and Technology of China. 3.3. Substrate binding site

2.5. Enzymatic activity assay To further characterize the substrate binding site, we obtained the structure of D-mannonate bound EcManD by quick soaking The catalytic kinetic parameters of wild-type of EcManD and its the crystal in 0.2 M D-mannonate. As shown in Fig. 2A, D-manno- mutants were measured according to a published procedure (Kim nate is coordinated in EcManD by a network of hydrogen bonds and Lee, 2008). Brieﬂy, EcManD activity was determined at 310 K and coordinate bonds. All the oxygen atoms of D-mannonate gener- by monitoring the formation of the complex of thiobarbituric acid ate hydrogen bonding interactions with EcManD residues includ- and the reaction product 2-KDG. The assay mixture contained ing Arg6, Glu63, Asn103, Asp109, His233, Asp236, Ser274, increasing concentrations of D-mannonate, 200 mM Tris–HCl buf- His298, Arg300, Arg349, Asp351, His352 and Tyr368. Also water fer at pH 7.5, and 1 lM EcManD. All measurements were per- mediated hydrogen bonds are found in the substrate binding site formed three times. The initial velocity versus substrate involving three waters and their associated residues Arg6, Ser64, concentration was analyzed by direct ﬁtting of data points to the Asn103, Asp109 and Gly183. Consistent with the previous result Michaelis–Menten equation using Origin software. (Zhang et al., 2009), the Mn2+ ion is present at the active site to associate with D-mannonate through coordinate bonds involving 3. Results and discussion one carboxyl oxygen atom and C2-hydroxyl (Fig. 2A). Unexpectedly, the D-mannonate in our EcManD-substrate struc- 3.1. Overall structure and the oligomerization state ture presents a different conformation with respect to that in the SsManD-substrate structure (Fig. 2B). Similar to those in Similar to other bacterial ManDs, the core structure of EcManD SsManD-substrate structure, EcManD residues Arg6, His233, is a TIM (ba)8 barrel, which is composed of strands b1–4, 7–9 and Asp236, His298, Arg349, Asp351, His352 and Tyr368 participate 12 and helices a1, 2, 4, 9, 10 and 12–14 (Fig. 1A). Besides the TIM in the coordination of D-mannonate. However, residues Glu63, barrel, a cap domain is present at the top of EcManD and the active Ser64, Asn103 and Asp109, whose counterparts do not make con- site resides in the interface between the TIM barrel and the cap tacts with D-mannonate in SsManD-substrate structure, contribute domain (Fig. 1A). Two a helices (a6 and a7) are formed by the in- to the immobilization of D-mannonate in EcManD-substrate struc- sert sequence of EcManD as compared to Gram positive ManDs. ture by forming hydrogen bonds with C5-hydroxyl and C6-hydro- In contrast to SsManD and EfManD (ManD from Enterococcus xyl of D-mannonate (Fig. 2A). In contrast, the EcManD residues faecalis), two Gram positive ManDs, forming dimers in solution, Ec- Tyr8 and Trp110 act conversely with respect to the corresponding ManD acts as a monomer (Fig. S1A). A series of conserved residues residues in SsManD (Fig. 2B). Of particularly, all of these substrate 330 X. Qiu et al. / Journal of Structural Biology 180 (2012) 327–334

Fig.1. (A) Cartoon diagram of the EcManD monomer. Two insert a helices of EcManD compared to Gram positive ManDs are highlighted in yellow. Loop1 and loop2 are highlighted in black. The associated D-mannonate is shown as cyan stick. The location of the substrate binding pocket is indicated with a dashed circle. (B) Overall view of the EcManD molecules in the asymmetric unit. Each monomer is shown in different color. (C) Structure-based multiple sequence alignment of the regions around the insert sequences of ManDs from Gram negative bacteria (top three) and the corresponding regions of those from Gram positives (bottom four). The secondary element assignment above the sequences corresponds to the structure of EcManD. The alignment was generated using the CLUSTALW web-server (Thompson et al., 2002).

binding residues in bacterial ManDs are conserved in primary se- ing, therefore, the D-mannonate in our EcManD-substrate structure quence and could align well in three dimensional space (Fig. 2C). may present an intermediate state of substrate entering, which is Similar binding sites but different substrate conformations in the then transformed to the SsManD-formed structure that is ready structures of EcManD-substrate and SsManD-substrate may reﬂect to be catalyzed. Structural comparison of D-mannonates in struc- the different stages of D-mannonate in binding to ManD. To evalu- tures of EcManD-substrate and SsManD-substrate (Fig. 2B) indi- ate the functional importance of the observed residues in the bind- cates that after entering the active site, a rotation around an axis ing site, we carried out enzymatic assays by using the various of the covalent bond between C2 and C3 is required to transform mutants. the D-mannonate into a conformation to be catalyzed, as that in As illustrated in Table 2, both the EcManDW110F and EcMan- SsManD-substrate structure, which is conﬁrmed by the result of DE63A/S64A mutants lose the catalytic activity. However, EcManDW110F mutant enzymatic test. EcManDN103A/D109A mutant retains a partial enzymatic activity (about

13%) due to a reduced kcat value as compared to wild-type, suggesting 3.4. Loop2 is above the active site and impacts the substrate entering these mutations impact the catalytic rate but not the substrate rate binding afﬁnity (a comparable Km value with that of wild-type). The kinetic data of EcManDE63A/S64A and EcManDN103A/D109A mu- Interestingly, the EcManD structure reveals that the insert se- tants suggest that Glu63 and Ser64 are essential for transferring quence, which is absent in Gram positive ManDs, forms two heli- the substrate to the active site, and Asn103 and Asp109 are also ces, a6 and a7, positioning above the active site (Fig. 1A). The the residues responsible for the substrate transferring process. a6, a7 and the linker loop, forming a V shaped motif, surround Mutations of Asn103 and Asp109, therefore, only alter the catalytic the a5 helix and make hydrophobic interactions involving rate. The reason for EcManDN103A/D109A mutant possessing residual Phe133, Phe136, Ile140, Leu141, Leu173, Ile177, Ile178 and activity is probably that Asn103 and Asp109 are positioned further Leu190, as well as a pi–pi conjugation between His139 and away from the simulated substrate entering tunnel analyzed with Phe162 (Fig. 3A). The relative orientations of a6 and a7 lead to a CAVER (Medek et al., 2007) as compared to Glu63 and Ser64 different conformation of the following loop which is immediately (Fig. 2D). Consistently, Glu63 and Ser64 are highly conserved in N-terminal to the a8 helix (termed as loop2) as compared to those bacterial ManDs, while the conservation of Asn103 and Asp109 is in Gram positive ManDs. In contrast to the absence of electron den- slightly lower (Fig. S3). sity of the corresponding loops of loop2 in both EfManD and Considering that the SsManD-substrate structure was obtained SsManD, the electron density of loop2 in EcManD is clear by co-crystallization (Zhang et al., 2009), but ours by crystal soak- (Fig. S4). Lacking of the insert sequence renders the loop2 (termed X. Qiu et al. / Journal of Structural Biology 180 (2012) 327–334 331

2+ Fig.2. (A) D-mannonate binding site of EcManD. D-mannonate and the interactive residues are shown in ball-and-stick models. Mn ion and water molecules are shown as 2+ purple and red spheres, respectively. Backbone of EcManD is shown in green ribbon model. The 2FO-FC electron density map covering D-mannonate and Mn ion contoured at 1.3r is represented as blue mesh. Detailed interactions between EcManD and D-mannonate are shown as close-up views in the right. Dashed red lines represent the potential 2+ hydrogen bonds. Dashed black lines represent the coordinate bonds of the Mn ion. (B) Structural alignment of the D-mannonates bound to EcManD and SsManD, which are shown in cyan and yellow ball-and-stick models, respectively. Backbones of EcManD and SsManD are shown in green and orange ribbon models, respectively. Tyr8, Trp110 of EcManD and SsManD are shown as side chains with ball-and-stick models. (C) Structural alignment of the substrate binding residues of EcManD and SsManD. The residues forming the substrate binding pocket in EcManD and SsManD are indicated and shown in green and orange sticks, respectively. (D) Details of D-mannonate entering tunnel of EcManD analyzed with CAVER shown as transparent surface with the D-mannonate and the simulated substrate entering track being in stick. Residues that are critical for substrate transfer are shown as side chains in stick models. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) according to EcManD) being away from the active site in SsManD lished by the insert sequence, is positioned on the top of the active (Fig. S5). However, the EcManD loop2, whose trajectory is estab- site. As shown in Fig. 3B, three hydrogen bonds are formed by res- 332 X. Qiu et al. / Journal of Structural Biology 180 (2012) 327–334

Table 2 Kinetic parameters for wild-type (wt) and mutants of EcManD measured at pH 7.5, 310 K.

wt E63A/S64A N103A/D109A W110F H32G N36G E185G E185D E186G E186D

Km (mM) 4.79±0.26 Inactive 4.08±0.13 Inactive 5.21±0.09 4.67±0.12 4.55±0.11 2.31±0.19 5.30±0.29 3.60±0.37 1 kcat (s ) 0.81±0.04 0.10 1.29±0.01 1.40±0.02 0.94±0.01 0.23±0.01 1.10±0.03 0.29±0.01

Fig.3. (A) Residues responsible for stabilizing a6 and a7 are indicated and shown as side chains in stick models. (B) Hydrogen bonds that immobilize loop2 are shown as red dashed lines. The hydrogen bonding residues are indicated and shown in stick models. The simulated substrate entering tunnel is shown as that in Fig 2D. (C) Comparison of the simulated D-mannonate entering tunnels of EcManD (green) and SsManD (orange). The tunnels are shown as transparent surface. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) idues Glu185, Glu186 in loop2 and His32, Asn36 from another loop The structural observations indicate that the substrate entrance (termed as loop1): Oe/Glu185Nd/His32, Oa/Glu185 and Oe/ locates between the side chains of Glu185 and Glu186 (Fig. 3B), Glu186side chain of Asn36. These hydrogen bonds help to keep loop2 could therefore regulate the EcManD activity with the the position of loop2, and appear to be essential to sustain the Glu185/Glu186-mediated interactions. The three hydrogen bonds structural integrity, as the EcManDE185G/E186G mutant, in which between loop1 and loop2 seem to bring them close to each other these hydrogen bonds are eliminated, tends to degrade into two and thereby narrow the substrate entering tunnel. If that were fragments. Loop2 together with loop1, forming a gorge outside the case, the EcManDH32G, EcManDN36G, EcManDE185G or EcMan- the catalytic pocket, seems to restrict the tunnels of substrate DE186G mutant would have a higher enzymatic activity than wild- entering and product releasing from the active site. Furthermore, type, as all of these mutations would disrupt the pre-existed CAVER calculations demonstrate that EcManD has a long and wind- hydrogen bond between loop1 and loop2 which in turn makes ing simulated substrate entering tunnel instead of a short and loop2 more flexible and consequently increases the radius of sub- straight one as in SsManD (Fig. 3C). Thus, we expected the catalytic strate entering tunnel. In contrast, respective substitution of rate of EcManD to be much lower than that of SsManD. Glu185 or Glu186 with Asp185 or Asp186 would shorten the side X. Qiu et al. / Journal of Structural Biology 180 (2012) 327–334 333 chain, but retains the carboxyl group. In such case, Asp185 or positives. Consistently, Gram negative bacteria have evolved ManDs Asp186 could still maintain the hydrogen bond with His32 or with low activities, and sequence insertion which reduces the activ- Asn36, respectively, leading to the loop2 being closer to loop1 than ities of Gram negative ManDs as compared to Gram positive ones is that in wild-type, thereby a decreased radius of substrate entering therefore deduced as one of the possible mechanisms leading to dif- tunnel is induced. ferent cell wall peptidoglycan content between these organisms on To test our structural analysis, the enzymatic activity assays of the level of metabolic status. these mutants were conducted, results of which are listed in Table 2. Consistent with the above hypothesis, all the EcManDH32G, 4. Conclusions EcManDN36G, EcManDE185G and EcManDE186G mutants exhibited higher activity as compared to wild-type. Notably, all the mutants This study reports the structures of EcManD and its complex present similar K values as that of wild-type, but larger k val- m cat with substrate, and reveals that the insert sequence of EcManD ues, implying not the substrate binding affinity but the catalytic as compared to Gram positive ManDs stabilizes a loop covering rate is altered by these mutations. The k value of EcManDN36G cat the substrate binding pocket. And the loop lowers the catalytic rate mutant is slightly higher than those of EcManDH32G, EcManDE185G demonstrated by comparative structural analysis in conjunction and EcManDE186G mutants, and the basis of this observation is with enzymatic activity assays. These features are conserved in probably that Asn36 forms two hydrogen bonds with loop2, so Gram negative ManDs. ManD participates in the Entner–Doudoroff the N36G mutation could disrupt these hydrogen bonds and make pathway using D-glucuronate as the substrate, thereby is related to loop2 more flexible than those in EcManDH32G, EcManDE186G and peptidoglycan biosynthesis when bacteria are grown on D-glucuro- EcManDE185G mutants. As expected, both the EcManDE185D and Ec- nate. The insertion of this sequence could decrease the D-glucuro- ManDE186D mutants show reduced enzymatic activities with com- nate metabolic rate and consequently lower the production of parable K values but significantly decreased k values as m cat precursor of peptidoglycan in Gram negative bacterium as com- compared to wild-type. pared to Gram positive ones. Therefore, this insert sequence may Taken together, these results demonstrate that the insert se- have functional importance that correlates with the difference of quence in EcManD causes a specific loop2 conformation, which cell wall peptidoglycan thickness between Gram positive and neg- influences the enzymatic activity by restricting the substrate ative bacteria. Moreover, substrate binding site analysis combining entering tunnel. with enzymatic activity assays demonstrates that some residues are essential for substrate to enter the active site and the substrate 3.5. Implications of the insert sequence functioning in carbohydrate requires a conformation transformation before catalysis. metabolism

All the structural and enzymatic information indicates that the 5. Accession numbers insert sequence in EcManD regulates the catalytic rate by control- ling the substrate entering tunnel. By comparison with the kinetic Atomic coordinates and structure factors of EcManD apo-form data of SsManD, we found that substrate affinity between SsManD and EcManD complexed with D-mannonate were deposited in the and EcManD do not change significantly, reflected by their similar Protein Data Bank (http://www.rcsb.org) under the accession 1 Km values. However, the kcat value of SsManD (5.88 s ) is about 6- codes 4EAC and 4EAY, respectively. fold higher than that of EcManD, implying that the insert sequence in EcManD lowers the catalytic rate. Moreover, sequence align- Acknowledgments ment demonstrates that His32, Asn36, Glu185, Glu186 and residues contributing to stabilizing a6 and a7 are conserved in Gram We would like to thank the staffs of SSRF for their assistance in negative ManDs with contrast that those residues are not con- data collection. Financial support for this work was provided by reserved in Gram positive ones (Fig. S3), which suggests that the sim- search grants from the Ministry of Science and Technology of P R ilar immobilization mode of loop2 (termed according to EcManD) China (Grant Nos.: 2012CB917200 and 2009CB825500) and the as EcManD exists in all Gram negative ManDs. Therefore, decreas- Chinese National Natural Science Foundation (Grant Nos.: ing the catalytic rate is a general function of the insert sequences in 31130018, 31170726) and Natural Science Foundation of Anhui Gram negative ManDs. Province (Grant No.: 090413085). In an anaerobic environment such as the intestine, bacteria utilize D-glucuronate as the sole carbon source to synthesize all carbohydrate components including peptidoglycan, which is the major Appendix A. Supplementary data structural component of bacterial cell wall and constitutes a consid- erable part of polysaccharides in bacterial cell (Hayhurst et al., Supplementary data associated with this article can be found, in 2008; Sorbara and Philpott, 2011). In this case, the products of the the online version, at http://dx.doi.org/10.1016/j.jsb.2012.06.013. Entner–Doudoroff pathway (pyruvic acid and glyceraldehyde 3-phosphate) could generate fructose-6-phosphate via gluconeo- References genesis (Nelson and Cox, 2000). Fructose-6-phosphate consequently produces UDP-N-acetylglucosamine and UDP-N-acet- Barreteau, H., Kovac, A., Boniface, A., Sova, M., et al., 2008. Cytoplasmic steps of ylmuramic acid, which are the donors of carbohydrate residues of peptidoglycan biosynthesis. FEMS Microbiol. Rev. 32, 168–207. peptidoglycan (Barreteau et al., 2008). Therefore, decrease of Collaborative Computational Project 4 1994. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763. catalytic rate of the enzyme participating in the Entner–Doudoroff DeLano, W.L., 2008. The PyMOL molecular graphics system. DeLano Scientific, San pathway could result in a low level of precursors for peptidoglycan Carlos, CA, USA. synthesis during bacterial cell growth and division when bacteria Dreyer, J.L., 1987. The role of iron in the activation of mannonic and altronic acid hydratases, two Fe-requiring hydro-lyases. Eur. J. Biochem. 166, 623–630. are grown on this gluconeogenic carbon source. The thickness of Emsley, P., Cowtan, K., 2004. Coot: model-building tools for molecular graphics. the peptidoglycan layer in cell wall of Gram negative bacteria is Acta Crystallogr. D Biol. Crystallogr. 6, 2126–2132. thinner than that in Gram positives by several-fold (Reith and Evans, P., 2006. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82. Mayer, 2011), which suggests that Gram negative bacteria probably Hayhurst, E.J., Kailas, L., Hobbs, J.K., Foster, S.J., 2008. Cell wall peptidoglycan need fewer carbohydrate precursors of peptidoglycan than Gram architecture in Bacillus subtilis. Proc. Nat. Acad. Sci. U.S.A. 105, 14603–14608. 334 X. Qiu et al. / Journal of Structural Biology 180 (2012) 327–334

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