Simon J. Charnock

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

Enzyme-catalyzed β-elimination of sugar uronic acids, exemplified by the degradation of plant cell wall pectins, plays an important role in a wide spectrum of biological processes ranging from the recycling of plant biomass through to pathogen virulence. The three-dimensional crystal structure of the catalytic module of a “family PL-10” polysaccharide lyase, Pel10Acm from Cellvibrio japonicus, solved at a resolution of 1.3 Å, reveals a new polysaccharide lyase fold and is the first example of a polygalacturonic acid lyase that does not exhibit the “parallel β-helix” topology. The “Michaelis” complex of an inactive mutant in association with the substrate trigalacturonate/Ca2+ reveals the catalytic machinery harnessed by this polygalacturonate lyase, which displays a stunning resemblance, presumably through convergent evolution, to the tetragalacturonic acid complex observed for a structurally unrelated polygalacturonate lyase from family PL-1. Common coordination of the −1 and +1 subsite saccharide carboxylate groups by a protein-liganded Ca2+ ion, the positioning of an arginine catalytic base in close proximity to the α-carbon hydrogen and numerous other conserved enzyme–substrate interactions, considered in light of mutagenesis data for both families, suggest a generic polysaccharide anti-β-elimination mechanism.

Promiscuity in ligand-binding: The three-dimensional structure of a Piromyces carbohydrate-binding module, CBM29-2, in complex with cello- and mannohexaose.

Carbohydrate–protein recognition is central to many biological processes. Enzymes that act on polysaccharide substrates frequently contain noncatalytic domains, “carbohydrate-binding modules” (CBMs), that target the enzyme to the appropriate substrate. CBMs that recognize specific plant structural polysaccharides are often able to accommodate both the variable backbone and the side-chain decorations of heterogeneous ligands. “CBM29” modules, derived from a noncatalytic component of the Piromyces equi cellulase/hemicellulase complex, provide an example of this selective yet flexible recognition. They discriminate strongly against some polysaccharides while remaining relatively promiscuous toward both β-1,4-linked manno- and cello-oligosaccharides. This feature may reflect preferential, but flexible, targeting toward glucomannans in the plant cell wall. The three-dimensional structure of CBM29-2 and its complexes with cello- and mannohexaose reveal a β-jelly-roll topology, with an extended binding groove on the concave surface. The orientation of the aromatic residues complements the conformation of the target sugar polymer while accommodation of both manno- and gluco-configured oligo- and polysaccharides is conferred by virtue of the plasticity of the direct interactions from their axial and equatorial 2-hydroxyls, respectively. Such flexible ligand recognition targets the anaerobic fungal complex to a range of different components in the plant cell wall and thus plays a pivotal role in the highly efficient degradation of this composite structure by the microbial eukaryote.

Key Residues in Subsite F Play a Critical Role in the Activity of Pseudomonas fluorescens Subspecies cellulosa Xylanase A Against Xylooligosaccharides but Not Against Highly Polymeric Substrates such as Xylan

In a previous study crystals of Pseudomonas fluorescens subspecies cellulosa xylanase A (XYLA) containing xylopentaose revealed that the terminal nonreducing end glycosidic bond of the oligosaccharide was adjacent to the catalytic residues of the enzyme, suggesting that the xylanase may have an exo-mode of action. However, a cluster of conserved residues in the substrate binding cleft indicated the presence of an additional subsite, designated subsite F. Analysis of the biochemical properties of XYLA revealed that the enzyme was a typical endo-β1,4-xylanase, providing support for the existence of subsite F. The three-dimensional structure of four family 10 xylanases, including XYLA, revealed several highly conserved residues that are on the surface of the active site cleft. To investigate the role of some of these residues, appropriate mutations of XYLA were constructed, and the biochemical properties of the mutated enzymes were evaluated. N182A hydrolyzed xylotetraose to approximately equal molar quantities of xylotriose, xylobiose, and xylose, while native XYLA cleaved the substrate to primarily xylobiose. These data suggest that N182 is located at the C site of the enzyme. N126A and K47A were less active against xylan and aryl-β-glycosides than native XYLA. The potential roles of Asn-126 and Lys-47 in the function of the catalytic residues are discussed. E43A and N44A, which are located in the F subsite of XYLA, retained full activity against xylan but were significantly less active than the native enzyme against oligosaccharides smaller than xyloseptaose. These data suggest that the primary role of the F subsite of XYLA is to prevent small oligosaccharides from forming nonproductive enzyme-substrate complexes.

Multifunctional xylooligosaccharide/cephalosporin C deacetylase revealed by the hexameric structure of the Bacillus subtilis enzyme at 1.9 Å resolution

Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities. Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C. The 1.9A structure of the multifunctional CE-7 esterase (hereinafter CAH) from Bacillus subtilis 168 reveals a classical alpha/beta hydrolase fold encased within a 32 hexamer. This is the first example of a hexameric alpha/beta hydrolase and is further evidence of the versatility of this particular fold, which is used in a wide variety of biological contexts. A narrow entrance tunnel leads to the centre of the molecule, where the six active-centre catalytic triads point towards the tunnel interior and thus are sequestered away from cytoplasmic contents. By analogy to self-compartmentalising proteases, the tunnel entrance may function to hinder access of large substrates to the poly-specific active centre. This would explain the observation that the enzyme is active on a variety of small, acetylated molecules. The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues. Protein residues involved in catalysis are tethered by interactions with protein excursions from the canonical alpha/beta hydrolase fold. These excursions also mediate quaternary structure maintenance, so it would appear that catalytic competence is only achieved on protein multimerisation. We suggest that the acetyl xylan esterase (EC 3.1.1.72) and cephalosporin C deacetylase (EC 3.1.1.41) enzymes of the CE-7 family represent a single class of proteins with a multifunctional deacetylase activity against a range of small substrates.

Cellvibrio japonicus α-L-arabinanase 43A has a novel five-blade β-propeller fold

Cellvibrio japonicus arabinanase Arb43A hydrolyzes the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans. The three-dimensional structure of Arb43A, determined at 1.9 Å resolution, reveals a five-bladed β-propeller fold. Arb43A is the first enzyme known to display this topology. A long V-shaped surface groove, partially enclosed at one end, forms a single extended substrate-binding surface across the face of the propeller. Three carboxylates deep in the active site groove provide the general acid and base components for glycosidic bond hydrolysis with inversion of anomeric configuration.

Substrate specificity in glycoside hydrolase family 10: Tyrosine 87 and leucine 314 play a pivotal role in discriminating between glucose and xylose binding in the proximal active site of pseudomonas cellulosa xylanase 10A

The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes β1,4-linked xylans but exhibits very low activity against aryl-β-cellobiosides. The family 10 enzyme, Cex, fromCellulomonas fimi, hydrolyzes aryl-β-cellobiosides more efficiently than does Xyl10A, and the movements of two residues in the –1 and –2 subsites are implicated in this relaxed substrate specificity (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998)Biochemistry 37, 4751–4758). The three-dimensional structure of Xyl10A suggests that Tyr-87 reduces the affinity of the enzyme for glucose-derived substrates by steric hindrance with the C6-OH in the –2 subsite of the enzyme. Furthermore, Leu-314 impedes the movement of Trp-313 that is necessary to accommodate glucose-derived substrates in the –1 subsite. We have evaluated the catalytic activities of the mutants Y87A, Y87F, L314A, L314A/Y87F, and W313A of Xyl10A. Mutations to Tyr-87 increased and decreased the catalytic efficiency against 4-nitrophenyl-β-cellobioside and 4-nitrophenyl-β-xylobioside, respectively. The L314A mutation caused a 200-fold decrease in 4-nitrophenyl-β-xylobioside activity but did not significantly reduce 4-nitrophenyl-β-cellobioside hydrolysis. The mutation L314A/Y87A gave a 6500-fold improvement in the hydrolysis of glucose-derived substrates compared with xylose-derived equivalents. These data show that substantial improvements in the ability of Xyl10A to accommodate the C6-OH of glucose-derived substrates are achieved when steric hindrance is removed.

Cellvibrio japonicus alpha-L-arabinanase 43A has a novel five-blade beta-propeller fold.

Cellvibrio japonicus arabinanase Arb43A hydrolyzes the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans. The three-dimensional structure of Arb43A, determined at 1.9 Å resolution, reveals a five-bladed β-propeller fold. Arb43A is the first enzyme known to display this topology. A long V-shaped surface groove, partially enclosed at one end, forms a single extended substrate-binding surface across the face of the propeller. Three carboxylates deep in the active site groove provide the general acid and base components for glycosidic bond hydrolysis with inversion of anomeric configuration.

Characterization of Escherichia coli OtsA, a trehalose-6-phosphate synthase from glycosyltransferase family 20

The Ots gene cluster of Escherichia coli encodes the synthetic apparatus for the formation of alpha,alpha-1,1-trehalose, a non-reducing glucose disaccharide. The otsA gene encodes a trehalose-6-phosphate synthase, a glycosyltransferase which catalyses the synthesis of alpha,alpha-1,1-trehalose-6-phosphate from glucose-6-phosphate using a UDP-glucose donor. It has been classified into glycosyltransferase family GT-20 based upon amino-acid sequence similarities. The otsA gene has been cloned and recombinant protein overexpressed using a pET-based system in E. coli BL21 cells. The recombinant protein (MW approximately 54.7 kDa) is active and has been crystallized in two forms suitable for X-ray diffraction analysis. The first is orthorhombic, P2(1)2(1)2(1), with unit-cell parameters a = 104.1, b = 127.8, c = 179.9 A. Data for this form have been collected to 3.0 A resolution at the CLRC Daresbury Synchrotron Radiation Source. The second form has unit-cell parameters a = b = 141.9, c = 317.8 A and displays the apparent space group P4(2). These crystals diffract beyond 2 A resolution, but display merohedral twinning.

Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations

Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α‐helices, the α‐domain, and another predominantly composed of β‐sheets, the β‐domain. Simulation flexibility analyses indicate that processive exolytic cleavage of hyaluronan, by PL8 hyaluronate lyases, is likely to involve an interdomain shift, resulting in the opening/closing of the substrate‐binding cleft between the α‐ and β‐domains, facilitating substrate translocation. Here, the Streptomyces coelicolor A3(2) PL8 enzyme was recombinantly expressed in and purified from Escherichia coli and biochemically characterized as a hyaluronate lyase. By using X‐ray crystallography its structure was solved in complex with hyaluronan and chondroitin disaccharides. These findings show key catalytic interactions made by the different substrates, and on comparison with all other PL8 structures reveals that the substrate‐binding cleft of the S. coelicolor enzyme is highly occluded. A third structure of the enzyme, harboring a mutation of the catalytic tyrosine, created via site‐directed mutagenesis, interestingly revealed an interdomain shift that resulted in the opening of the substrate‐binding cleft. These results add further support to the proposed processive mechanism of action of PL8 hyaluronate lyases and may indicate that the mechanism of action is likely to be universally used by PL8 hyaluronate lyases. Proteins 2011.

Characterization of a novel pectate lyase, Pel10A, from Pseudomonas cellulosa

Biological recycling of plant material is essential for biosphere maintenance. This perpetual task involves a complex array of enzymes, including extracellular polysaccharide hydrolases and lyases. Whilst much is known about the structure and function of the hydrolases, relatively little is known about the structures and mechanisms of the corresponding lyases. To this end, crystals of the catalytic module of a novel family 10 pectate lyase, Pel10A from Pseudomonas cellulosa, were obtained using polyethylene glycol 2000 monomethylether as a precipitant. They belong to space group P2(1), with unit-cell parameters a = 47.7, b = 106.1, c = 55.4 A, beta = 92.0 degrees, and have two molecules in the asymmetric unit. The crystals diffract beyond 1.5 A using synchrotron radiation.