Convergent evolution sheds light on the anti- ␤-elimination mechanism common to family 1 and 10 polysaccharide

Simon J. Charnock*†, Ian E. Brown‡, Johan P. Turkenburg*, Gary W. Black‡, and Gideon J. Davies*§

*York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, United Kingdom; and ‡School of Applied Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, United Kingdom

Communicated by Perry A. Frey, University of Wisconsin, Madison, WI, July 19, 2002 (received for review April 9, 2002) -catalyzed ␤-elimination of sugar uronic acids, exemplified flava are responsible for the majority of fresh fruit and vegetable by the degradation of plant cell wall pectins, plays an important spoilage (7). The critical role of polygalacturonate lyases in plant role in a wide spectrum of biological processes ranging from the development is emphasized by the dedication of at least 34 ORFs recycling of plant biomass through to pathogen virulence. The for this function in Arabidopsis thaliana (8). three-dimensional crystal structure of the catalytic module of a The CAZy classification (5) describes 12 families of polysac- ‘‘family PL-10’’ polysaccharide , Pel10Acm from Cellvibrio charide lyases with polygalacturonate-active found in japonicus, solved at a resolution of 1.3 Å, reveals a new polysac- families PL-1, 2, 3, 9, and 10. Family PL-10 currently comprises charide lyase fold and is the first example of a polygalacturonic acid just seven sequences and is a family for which no structural or lyase that does not exhibit the ‘‘parallel ␤-helix’’ topology. The mechanistic data exist. Here we report the 1.3-Å resolution ‘‘Michaelis’’ complex of an inactive mutant in association with the three-dimensional structure of the competent catalytic module substrate trigalacturonate͞Ca2؉ reveals the catalytic machinery of the polygalacturonic acid lyase Pel10A (Pel10Acm), from harnessed by this polygalacturonate lyase, which displays a stun- Cellvibrio japonicus, together with analysis of the activity of ning resemblance, presumably through convergent evolution, to wild-type and mutant enzymes. The enzyme topology reveals a ␣ ␣͞␣ the tetragalacturonic acid complex observed for a structurally predominantly -helical enzyme with a distorted ( )3 barrel unrelated polygalacturonate lyase from family PL-1. Common co- quite unlike the parallel ␤-helix displayed by other pectate lyases ordination of the ؊1 and ؉1 subsite saccharide carboxylate groups (Pel). The ‘‘Michaelis complex’’ of an inactive mutant of Pel10A 2؉ by a protein-liganded Ca ion, the positioning of an arginine with the substrate trigalacturonic acid GalA3 reveals the catalytic catalytic base in close proximity to the ␣-carbon hydrogen and machinery and supports via an E1cb or concerted E2 numerous other conserved enzyme–substrate interactions, consid- elimination mechanism with Brønsted base catalysis provided by ered in light of mutagenesis data for both families, suggest a arginine. The active center provides a stunning example of generic polysaccharide anti-␤-elimination mechanism. convergent evolution. The location of three substrate-binding 2ϩ

arginines, a main-chain carbonyl-O3 interaction, the Ca co- BIOCHEMISTRY olysaccharide lyases (EC 4.2.2.x) are carbon–oxygen lyases ordinating carboxylates and the potential Brønsted base itself are Pthat harness ␤-elimination chemistry (reviewed in ref. 1) to isostructural with the catalytic center of the totally unrelated bring about degradation of C5 uronic acid containing pyranoside family PL-1 enzyme Pel1C from E. chrysanthemi. substrates such as polygalacturonates, alginates, hyaluronan, and Materials and Methods chondroitin. They play a pivotal role in a wide range of processes ranging from the recycling of plant material, a process essential Production of SeMet and Native Protein. Protein production and for biosphere maintenance (2), through to virulence of patho- purification were achieved essentially as described (9) except the gens (3, 4). In contrast to the 87 sequence-derived families of methionine auxotroph Escherichia coli B834 (DE3, Novagen), glycoside , polysaccharide lyases have been classified transformed with p4.2.1 (10), was used for both native and into just 12 families on the basis of amino acid sequence SeMet preparations. Matrix-assisted laser desorption ionization- similarities (5), reflecting the requirement for substrate uronic time of flight mass spectrometric analysis of native and SeMet- acid groups in the elimination mechanism. Three-dimensional containing protein confirmed the identity of the polypeptides structures have been reported for enzymes from polysaccharide and indicated that the N-terminal methionine residue had been lyase (PL) families 1, 3, 5, 6, 8, and 9 and have thus far revealed processed by the host bacterium (data not shown). The ␣͞␣ QuikChange Site-Directed Mutagenesis kit (Stratagene) was just two catalytic module topographies: the ‘‘( )6’’ barrel seen in families PL-5 and 8, or the ‘‘parallel ␤-helix’’ revealed by the used to mutate plasmid p4.2.1. first structure determination for a polysaccharide lyase, that of Kinetic Analyses. Pel1C from family PL-1, and observed subsequently in structures Kinetic parameters were determined for native from families PL-3, 6, and 9. A catalytic mechanism featuring and derivative forms of Pel10Acm against Na-polygalacturonic ϩ acid and trigalacturonic acid (Sigma-Aldrich). Release of the proton abstraction from C5 of the 1 subsite sugar residue, ␣ termed the ␣-carbon, and proton donation to the glycosidic 4-deoxy- -D-gluc-4-enuronosyl-containing products was fol- oxygen, with the elimination of the leaving group from C4, lowed on a Helios Alpha UV-visible spectrometer (Thermo- termed the ␤-carbon (1, 6) seems the most plausible. Spectronic) at 232 nm, with a 1-cm light path quartz cuvette. The reaction mixture, 0.5 ml, comprised substrate in 50 mM CAPS Polygalacturonic acid lyases (EC 4.2.2.2; polygalacturonate transeliminases) are extracellular enzymes found in plants and also secreted by both pathogenic and saprophytic microorgan- ␣ Abbreviations: GalA, galacturonic acid; Pel, pectate lyase; PL, polysaccharide lyase. isms. They cleave polymeric -1,4-linked galacturonic acids Data deposition: The coordinates and observed structure factor amplitudes for the struc- (GalA) generating 4,5-unsaturated oligogalacturonates as prod- tures described in this paper have been deposited in , www.rcsb.org [PDB ucts (6). In addition to their role in the carbon cycle, polygalac- ID codes: 1gxm (P21 native), 1gxn (P21212 native), and 1gxo (P21212 GalA3 complex)]. turonic acid lyases are important virulence factors of plant †Present address: School of Applied Sciences, Northumbria University, Newcastle upon Tyne pathogens, such as Erwinia chrysanthemi (3), whereas the en- NE1 8ST, United Kingdom. zymes from Pseudomonas fluorescens and Pseudomonas viridi- §To whom reprint requests should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.182431199 PNAS ͉ September 17, 2002 ͉ vol. 99 ͉ no. 19 ͉ 12067–12072 Downloaded by guest on September 28, 2021 buffer, pH 10.0, containing calcium chloride at a concentration Results of 2 mM (with GalA3 as substrate) or 0.1 mM (with polygalac- Pel10A from Cellvibrio japonicus comprises an N-terminal car- turonic acid as substrate; the viscosity of polygalacturonic acid ϩ bohydrate-binding module (CBM family 2a), a central X4 mod- in high [Ca2 ] preventing utilization of higher concentrations). ule of unknown function, and a C-terminal polygalacturonic acid The reaction components were prewarmed to, and the assay lyase catalytic module classified into family PL-10 (10). This performed at, 310 K. Individual kinetic parameters were calcu- C-terminal module (residues 327–649) had previously been lated by using GRAFIT VERSION 4 (Erithacus Software, Surrey, expressed as a separate entity, termed Pel10Acm, and shown to U.K.). To investigate bond cleavage frequencies by using tri- and be an endo-acting polygalacturonic acid lyase with activity solely tetragalacturonic acid, substrate consumption and product ap- against the homogalacturonic acid backbone. Catalytic activity is pearance were followed over time by using high-pressure anion- optimal at pH 10.3 and is absolutely dependent on Ca2ϩ with exchange chromatography as described (10) with the unsatur- maximal activity at Ϸ2mM[Ca2ϩ] (10), as observed for many ated nonreducing end of the product used to establish the other polysaccharide lyases (6, 18–20) and supported by three- location of the scissile bond. dimensional analysis of enzymes from family PL-1 (3, 6, 21–23). Quantitative analysis of the kinetics of Pel10Acm, after the Crystallization, Data Collection, and Processing. Native and SeMet release of the unsaturated products from polygalacturonate Pel10Acm crystals were grown as described (9). Crystals belong degradation yields kcat and KM for the wild-type enzyme of 408 ϭ ϭ Ϫ Ϫ to space group P21, with unit cell dimensions a 47.9, b 106.7, s 1 and 0.074 mg ml 1, respectively. Pel10Acm had no detectable ϭ ␤ ϭ c 55.6 Å, 92.0° and have two molecules in the asymmetric activity against GalA2. Against GalA3, the enzyme exclusively unit. Native crystals grown in the presence of 25 mM CaCl2 and cleaved the substrate in the Ϫ1toϩ2 subsite-binding mode mutant D389A crystals cocrystallized with 20 mM GalA3, belong (nomenclature according to ref. 24), releasing galacturonic acid Ϫ1 to closely related space group P21212, with unit cell dimensions and the 4,5-unsaturated GalA2 with a kcat and KM of 1,075 s ϭ ϭ ϭ Ϫ a 106.3, b 55.2, c 47.7 Å, and have one molecule in the and 0.6 mM, respectively. GalA4 was cleaved both in the 2to asymmetric unit. Before data collection a rayon-fiber loop was ϩ2 (64%) or the Ϫ1toϩ3 (36%) subsite-binding modes but the used to transfer single crystals to a cryoprotectant solution scarcity of tetrasaccharide substrate prevented determination of ͞ consisting of the growth buffer supplemented with 25% (vol vol) accurate kinetic parameters for GalA4. glycerol. A three-wavelength MAD experiment, at 100 K, was con- Three-Dimensional Structure of Pel10Acm. The three-dimensional ducted on beamline BM14 at the European Synchrotron Radi- structure of Pel10Acm was solved at a resolution of 1.65 Å (see ation Facility at Grenoble, France, with a MAR CCD detector. Materials and Methods). Native data allow the resolution to be The wavelengths for the MAD experiment were chosen by extended to 1.32 Å. The three-dimensional structure (Fig. 1a), scanning through the absorption edge of the Se-Pel10Acm extending from residues 339 to 648, does not adopt the parallel crystal. Data sets were collected at the wavelength correspond- ␤-helix fold displayed both by polygalacturonic acid lyases from ing to the minimum ƒЈ (0.97930Å), the maximum ƒЈЈ (0.97889Å) families PL-1 (Fig. 1b), -3, and -9 and other enzymes active on and a reference wavelength at an energy above the absorption polygalacturonates. Instead, it is a predominantly ␣-helical struc- edge (0.88560Å) to maximize dispersive differences. Native data ture. The topography of the protein reveals two ‘‘domains’’ the were collected on a single crystal belonging to space group P21, interface of which forms a wide central chasm, the location for at 100 K, on beamline ID14–4 at the European Synchrotron the substrate-binding and catalytic residues (Fig. 1c). The N- Radiation Facility, Grenoble; data for the native crystals in space terminal, helical domain is of the ␣-toroid form, displaying a ␣͞␣ group P21212 on station PX9.6 at the SRS, Daresbury, U.K.; and compact ( )3 barrel. The sixth helix (residues 479–487) of the data for the complex with GalA3 on beamline ID29 of the toroid crosses over to the N-terminal domain, which is predom- European Synchrotron Radiation Facility, Grenoble. All data inantly formed by irregular coil, a sheet of short ␤-strands, and were processed by using the HKL2000 suite of programs (11). a further three helices. The P21 crystal form contains two molecules that are extremely similar, except for a small rigid- Structure Solution. Unmerged data for the selenium-MAD ex- body closure of the N-terminal domain of molecule B compared periment were input to SOLVE (12) to locate 12 Se positions, with A. The segment from residues 368–381 shows the greatest corresponding to two molecules of Pel10Acm in space group P21. movement resulting in 3- to 4-Å shifts in the position of the side The Se positions were refined and phases calculated by using chains of Asn370 and Asp372, which are part of a loop that sites SOLVE. These phases were used as a starting set for phase above one wall of the substrate-binding cleft, described below, improvement, incorporating the native data initially at 1.6 Å with Asn370 some 5–7 Å distant from the trisaccharide ligand. resolution, with DM (13). ARP (14) and REFMAC (15) were used Although the predominantly ␣-helical structure of Pel10Acm to generate an electron density map which allowed tracing bares no relationship to other polygalacturonic acid lyases, of a single molecule with the X-AUTOFIT module in QUANTA limited topological similarity to other ␣-toroidal folds exists, ␣͞␣ ␣͞␣ (Accelrys, San Diego). The second molecule was located by including the ( )6 and ( )7 toroids displayed by other classes using AMORE (16). The two molecules were refined with of carbohydrate-active enzymes. Glycoside hydrolases from fam- REFMAC, initially with the phases from DM included as exper- ily GH-47, ␣-1,2-mannosidases (for example, Protein Data Bank ␣͞␣ imental restraints. The final model has a crystallographic R code 1hcu), possess an ( )7 topology six of whose helices ϭ factor of 0.130 (Rfree 0.162) and has 98% of residues in correspond well with Pel10Acm with 231 C␣ atoms overlapping ␣͞␣ ‘‘allowed’’ regions of the Ramachandran plot [calculated by with rms 3.2 Å. The ( )6 barrels of other glycoside hydrolases using MOLEMAN2 (17)]. One molecule from the native structure including family GH-8 cellulases, GH-15 glucoamylases, and in space group P21 was used as a starting model for refinement family GH-65 maltose phosphorylases are also similar, as are in space group P21212, because the unit cells are very closely some sugar epimerases, such as GlcNAc 2-epimerases (PDB 1fp3 related. After 10 initial cycles of rigid body refinement, individ- shares 227 overlapping C␣ atoms with rms 3.4 Å). Many ual coordinates and temperature factors were refined as above. polysaccharide lyases acting on other uronic acid polymers, ϭ The final model has a crystallographic R factor of 0.129 (Rfree including the family PL-5 alginate and PL-8 chondroitin and ␣͞␣ 0.157). The P21212 native model was used as the starting model hyaluronan lyases, also display variants on the ( )6 fold, which ␣ for the complex with GalA3 which, with the same Rfree reflec- suggests that the -toroid fold, in its many guises, is a powerful tions, has a crystallographic R factor of 0.17 and Rfree 0.23. and adaptable scaffold for carbohydrate-active enzymes upon

12068 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.182431199 Charnock et al. Downloaded by guest on September 28, 2021 Fig. 1. (a) Three-dimensional structure of the catalytic domain of Pel10 in divergent (wall-eyed) stereo; (b) Pel1C ligands and the potential base arginine residues in ‘‘ball-and-stick’’ representation, with the Ca2ϩ ions as shaded spheres, and the structures color-ramped from N to C terminus [figure prepared by using ϩ MOLSCRIPT͞BOBSCRIPT (32, 33)]. (c) Divergent (wall-eyed) stereo surface representation of Pel10Acm with the ligand as licorice, the Ca2 as a sphere, and the guanidinium group of Arg524 shaded blue (prepared by using GRASP 34). BIOCHEMISTRY which a diverse array both of specificities and catalytic mecha- Glu535. Mutation of Glu535 indeed reduces activity approxi- nisms may be grafted. mately 200-fold even on GalA3 from which it lies some 7–10 Å distant, suggesting that it does indeed play a role in maintaining 2؉ GalA3͞Ca Complex of Pel10Acm and Site-Directed Mutagenesis at the structural integrity of the catalytic center groups. the Catalytic Center. Cocrystallization of native Pel10Acm with In the ϩ1 subsite, where the enzyme chemistry occurs, Arg625 substrates revealed neither ligand nor Ca2ϩ (data not shown). An forms a hydrogen-bonding interaction with both the C2 and C3 inactive mutant, D389A, was used to obtain a ‘‘Michaelis’’ hydroxyl groups of the ϩ1 galacturonic acid moiety, reminiscent 2ϩ͞ complex with Ca GalA3 to a resolution of 2.15 Å. Continuous of a similar interaction observed in family 11 xylanases (27). O3 electron density for the trisaccharide substrate, each sugar unit makes a further interaction with the main-chain carbonyl of 4 present in its C1 (chair) conformation, and additional density Gly628 (discussed below). The side chain of Arg524 is positioned relating to an active-site-liganded Ca2ϩ were clear (Fig. 2). The 2.5 Å from the hydrogen atom on C5 of the galacturonic residue trisaccharide occupies subsites Ϫ1toϩ2, consistent with the at subsite ϩ1, stabilized through two hydrogen bonds with unique mode of bond cleavage for this substrate, and allows Glu527. Arg524 is thus the only potential catalytic base in close description of the enzyme–substrate interactions. proximity to H5 in the ϩ1 subsite. The side-chain hydroxyl group At the Ϫ1 (leaving-group) subsite, Arg596 forms a salt bridge of Tyr526 is a hydrogen bond donor to the ϩ2 sugar carboxylate, with the substrate carboxylate group, which in turn also forms a whereas Arg610 forms a salt bridge with this group. The coordinate bond with the Ca2ϩ ion (Fig. 3). The R596A deriv- involvement of the Ϫ1 and ϩ2 subsite carboxylate groups in Ϫ ϩ ative displayed very little detectable activity against GalA3 salt-bridge formation, and the coordination of 1 and 1 subsite although it maintained Ϸ0.5% activity against polymeric sub- carboxylate oxygen atoms with Ca2ϩ, may contribute to the strates (Table 1). The deleterious effects of some mutations specificity of Pel10A for homopolygalacturonic acid. At the ϩ1 being mitigated by the additional binding energy derived from subsite, the majority of mutations result in complete loss of long polymeric substrates are a common feature in polysaccha- catalytic activity. D389A, R524A, R524K, E527A, and E527Q 2ϩ ride degradation (see, for example, refs. 25 and 26). The Ca ion derivatives were all inactive on both GalA3 and polygalacturonic is also coordinated by an oxygen atom from the ϩ1 subsite sugar acid. Mutation of Asn390 to Ala resulted in 20- to 100-fold carboxylate and one carboxylate oxygen atom from Asp451 with reductions in activity on polygalacturonate and GalA3 respec- three water molecules completing hexacoordination. Substitu- tively. R625A and R625K mutations resulted in almost total loss tion of Asp451, the only close protein ligand of the Ca2ϩ ion, led of activity against small soluble substrates, with substantially ϩ to a total loss of activity on both GalA3 and polygalacturonic acid reduced catalytic efficiency against polygalacturonate. In the 2 (Table 1). The side chain of Asp451 lies at the end of a helical subsite, the Y526F mutation led to 2- to 3-fold reductions in segment that is held in appropriate main-chain conformation specific activity on both polygalacturonic acid and GalA3 through direct hydrogen bonds from both its flanking main- whereas the R610A mutant is five times less active against chain amide groups through to the side-chain carboxylate of polymeric substrates but 25 times less active against the trisac-

Charnock et al. PNAS ͉ September 17, 2002 ͉ vol. 99 ͉ no. 19 ͉ 12069 Downloaded by guest on September 28, 2021 2ϩ Fig. 2. Observed electron density for the GalA3͞Ca complex of Pel10Acm. The map shown is a maximum-likelihood͞␴A weighted 2Fobs Ϫ Fcalc syntheses contoured at 0.33 electrons per Å3. The Pel10A structure is in yellow and the convergent evolution of unrelated pectate lyases revealed by the active-center overlap of R218K mutant of Pel1C shown in dark green. (Arg218 has been reintroduced, in its native location, for reference.) The relative locations of the two putative catalytic base arginines within the protein framework are shown in Fig. 1 a and b for Pel10 and Pel1, respectively.

charide. The catalytic inactivity of the D389A mutant, located spite no topological similarity between these enzymes (Fig. 1 a between the ϩ1 and ϩ2 subsites, is superficially surprising, given and b). In both cases substrate carboxylates at the putative Ϫ1 that the mutant is isomorphous with the native structure and that and ϩ1 subsites are coordinated with a Ca2ϩ ion that is in turn this residue lies some 5–7 Å from the nearest atom of the liganded to a strictly conserved acidic amino acid; Asp451 in substrate. This inactivity is discussed further below in light of the Pel10 and Glu166 in Pel1C. The interactions of the ϩ1 subsite similarity with other pectate-active enzymes. sugar are identical including a main-chain carbonyl interaction with O3 and an arginine interaction with both O3 and O2. An Discussion identical location also exists for the putative catalytic base Evidence for Convergent Evolution of Catalytic Mechanism. Pel10- Arg524 of Pel10A with the well characterized base of Pel1C, Acm reveals a stunning example of the convergent evolution of Arg218. Asp162 of Pel1C, which coordinates a second calcium ͞ 2ϩ catalytic mechanism. Comparison of the Pel10Acm GalA3 Ca ion to the ϩ1 sugar carboxylate, finds no direct equivalent in the ͞ 2 complex with the inactive mutant R218K GalA4 Ca complex of D389A mutant of Pel10A. This position, however, is isostruc- Pel1C (kindly provided by F. Jurnak) (6) (Fig. 2), reveals an tural with the position of the Asp389 side chain of native Pel10A essentially identical disposition of six active-center groups de- and a similar role for this residue may be envisaged. The trisaccharide itself is also found in an essentially identical Ϫ ϩ conformation to the 1to 2 subsites of the GalA4 complex of Pel1C, and the structural similarity even extends to the ϩ2 subsite where the only close interaction of Pel10A, that from the carboxylate of the substrate to Arg610, is also invariant in Pel1C. Both Pel10Acm and Pel1 perform anti ␤-elimination charac-

Table 1. Relative activities for native and mutant forms of Pel10Acm

Polygalacturonic acid GalA3

Native 100% 100% D389A ND* ND N390A 5.7 1.1 D451A ND ND R524A ND ND R524K ND ND Y526F 48.9 30.5 E527A ND ND E527Q ND ND E535A 0.38 0.47 R596A 0.44 0.04 R610A 21.8 3.7 R625A 0.41 0.02 Fig. 3. Schematic diagram of the interactions of the mutant D389A R625K 3.32 0.14 Pel10Acm with trigalacturonate. The approximate location of Asp389 from Ϫ the native structure is indicated for reference. *, ND, not detectable, activity Ͻ0.05 s 1.

12070 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.182431199 Charnock et al. Downloaded by guest on September 28, 2021 BIOCHEMISTRY

Fig. 4. (a) More O’Ferral diagram for ␤-elimination of galacto-configured uronic acids. (b) Putative E1cb͞asynchronous E2 reaction mechanism for Pel10 and related enzymes in which proton abstraction by arginine is followed by leaving-group elimination. The essential role of Asp389 may involve a role in binding a second Ca2ϩ ion as observed in Pel1C.

terized by Brønsted base-catalyzed abstraction of the H5 proton substrate C5 hydrogen atom is Arg524, located at the putative (acidified by the electronegative carboxylate substituent at C5) ϩ1 subsite (Figs. 2–4), which is in a location identical to the and elimination of the substituent at O4 generating a 4,5- putative arginine base of PL-1 enzymes (6). We therefore unsaturated product (Fig. 4). The mechanistic landscape for this conclude that a deprotonated arginine functions as the catalytic reaction is best considered in light of the More O’Ferrall diagram base in both systems which, given the pH optima of about 10.5 (Fig. 4a), in which the courses of the three pathways, referred to this function represents only a 2-unit pKa perturbation for this as E1, E2, and E1cb, are determined by the order of bond arginine, similar to the well characterized pKa shifts of carbox- cleavage (1). In the E1-type reaction, the C␤OX bond is cleaved ylate groups in glycoside hydrolases. Calculations on the Pel1C before C␣OH, a stabilized carbocation intermediate formed system indeed revealed appropriate perturbation of the arginine followed by subsequent elimination of the proton from the pKa by virtue of its interactions with adjacent carboxylates (3). ␣-carbon; such reactions in aqueous solution or enzyme active In Pel10A Arg524 interacts with both substrate and side-chain sites are widely considered unlikely (1). Alternatively, the E1cb carboxylates. Arginine is a rare base in enzymatic reactions, the reaction involves a carbanion intermediate preceded by ␣-car- reverse reaction of quinol:fumarate reductase providing another bon proton abstraction with leaving-group elimination occurring example (29), but it is perhaps not surprising given the high pH subsequently. The E2 pathway reflects concerted proton ab- optima of pectate lyases. The superfamily operates at a straction and leaving-group elimination, although mechanistic much lower pH and instead utilizes lysine for proton abstraction boundaries may be rather blurred (28). The catalytic mechanism (30, 31). is further classified to reflect the location of the two leaving The notable absence of a classical Brønsted acid (either groups either on the same face of the incipient double bond (syn enzyme or substrate-derived) in proximity to the oxygen of the elimination) or on opposite sides (anti elimination). Crystallog- scissile glycosidic bond in both complexes suggests that little or raphy alone may not discriminate between these mechanistic no buildup of negative charge may occur at this center at the pathways but the active-center similarity of this enzyme, with rate-limiting transition state, which implies either an E1cb structurally unrelated anti-eliminases from family PL-1 and the reaction by a resonance-stabilized putative aci-acid carbanion apparent absence of any Brønsted acid in the vicinity of O4, intermediate (Fig. 4b) or an E2 reaction with concerted but allows some description of the likely reaction trajectory. asynchronous bond cleavage. In either case, elimination of the The only potential catalytic base in close proximity to a leaving group would not be wholly rate-limiting, and proton

Charnock et al. PNAS ͉ September 17, 2002 ͉ vol. 99 ͉ no. 19 ͉ 12071 Downloaded by guest on September 28, 2021 donation to the leaving group may subsequently be achieved tyrosine. The 12% residual activity for the H225A mutant of the through solvent water. The nearest group to either of the lone Streptococcus pneumoniae seems to rule out its pairs of the glycosidic oxygen is in fact the O3 hydroxyl of the ϩ1 role as a base, whereas mutation of the tyrosine residue results subsite sugar at 2.9 Å (but with C3OO3OO4 angle 55°). Given in a complete loss of activity (4). Complexes of family PL-8 the total structural invariance of the coordination of O3 in Pel1 enzymes do, however, identify a conserved arginine residue and Pel10, with arginine and main-chain carbonyl ligands, it may analogous in position to that of Pel10Acm, which lies in close be that leaving-group protonation is achieved from Arg625 in a proximity to the ␣-carbon of bound substrate (20). Cleavage of shuttle involving the O3 hydroxyl and main-chain carbonyl the ␤-glycosidic bonds of hyaluronan and chondroitin substrates moieties. It is also formally possible that Lewis acid assistance to with equatorial C4 substituents involves syn elimination, which is 2ϩ leaving-group departure, facilitated by Ca , plays a role, but no chemically more challenging. PL-5 and 8 enzymes need not metal ion seems appropriately positioned in either the PL-1 or necessarily share a similar catalytic mechanism to those from PL-10 enzyme families. PL-1 and 10 and may indeed feature a less concerted reaction The critical role played by Asp389 in catalysis is intriguing pathway (Fig. 4a). Many polysaccharide lyases play a role in the given the 5- to 6-Å distance between this residue and the nearest ͞ virulence of pathogens of eukaryotes. Hyaluronate lyases substrate atoms. The overlap with the inactive R218K GalA4 2ϩ spreading and infection factors for highly pathogenic bacteria Ca complex of Pel1C (6) reveals that each oxygen of the such as S. pneumoniae, for example. Polygalacturonic acid lyases ␣-carbon carboxylate in subsite ϩ1 of Pel1C is liganded to a ϩ themselves are involved in pathogenic infection of plants and separate Ca2 ion, suggestive of a role played by electrophilic foodstuff degradation. The design of inhibitors on the basis of catalysis and somewhat reminiscent of the divalent metal the likely transition state(s) for the E2͞E1cb reaction mecha- ␮-bridges observed for other eliminases such as the enolase nisms is, therefore, of great importance. The Pel10Acm struc- superfamily. Twin metal ions function both through the acidi- ture and its revealing active-center similarities with Pel1C begin fication of the adjacent ␣-proton and stabilization of the subse- quent aci-acid carbanion intermediate (30, 31). Given the mech- to shed light on the mechanistic conundrums and should, in anistic similarities observed between PL-1 and PL-10 enzymes, harness with appropriate physical-chemical studies, lead to the and the similar location of the carboxylate of Asp389 with design of anti-infective agents against both human and plant Asp162 of Pel1C, it is possible that the D389A mutant acts by way pathogens. of disruption of an analogous second Ca2ϩ-coordinating system Note Added in Proof. leading to complete loss of enzyme activity as is witnessed when Recent work has shown Pseudomonas fluorescens 2ϩ subsp. cellulosa, sometimes also described as Pseudomonas cellulosa,to the first Ca coordination sphere is mutated in the D451A be a Cellvibrio species (35, 36). It has been proposed that the organism mutant. be renamed Cellvibrio japonicus to reflect this fact. Despite the availability of both native and complexed three- dimensional structures of PL-8 chondroitin and hyaluronate This work was supported by the Higher Education Funding Council for lyases and corresponding mutagenesis data, no consistent cata- England, the Biotechnology and Biological Sciences Research Council, lytic mechanism exists. Two of the hypothesized mechanisms the Yorkshire Agricultural Society, and the Wellcome Trust. G.J.D. is a feature a histidine as the catalytic base, whereas others implicate Royal Society University Research Fellow.

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