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Convergent Evolution Sheds Light on the Anti- -Elimination Mechanism

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Convergent Evolution Sheds Light on the Anti- -Elimination Mechanism Convergent evolution sheds light on the anti- ␤-elimination mechanism common to family 1 and 10 polysaccharide lyases 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) Enzyme-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 lyase, Pel10Acm from Cellvibrio charide lyases with polygalacturonate-active enzymes 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 catalysis 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 hydrolases, 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 Protein Data Bank, 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),
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