Richard W. Pickersgill

Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities

Germin is a hydrogen peroxide generating oxalate oxidase with extreme thermal stability; it is involved in the defense against biotic and abiotic stress in plants. The structure, determined at 1.6 Å resolution, comprises β-jellyroll monomers locked into a homohexamer (a trimer of dimers), with extensive surface burial accounting for its remarkable stability. The germin dimer is structurally equivalent to the monomer of the 7S seed storage proteins (vicilins), indicating evolution from a common ancestral protein. A single manganese ion is bound per germin monomer by ligands similar to those of manganese superoxide dismutase (MnSOD). Germin is also shown to have SOD activity and we propose that the defense against extracellular superoxide radicals is an important additional role for germin and related proteins.

β-Glucosidase, β-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes wit 8-fold β/α architecture and with two conserved glutamates near the carboxy-terminal ends of β-strands four and seven

Comparison of the recently determined crystal structures Pseudomonas fluorescens subsp. cellulosa family F xylanase, (1–3)‐β‐glucanase and (1–3,1–4)‐β‐glucanase and the catalytic domain of E. coli β‐galactosidase reveals that they belong to a superfamily of 8‐fold β/α‐barrels with similar amino acid residues at their active sites. In the three families that these enzymes represent, the nucleophile is a glutamate, which is located close to the carboxy‐terminus of β‐strand seven. In addition all three enzymes have the sequence asparagine‐glutamate close to the carboxy‐terminus of β‐strand four. This glutamate has been identified as the acid/base in the family F xylanases and is essential for catalysis in β‐galactosidase. We suggest that the equivalent residue in the barley glucanases is the acid/base. Analysis of the sequences of family 1 β‐glucosidases and family 5 cellulases shows that these enzymes also belong to this superfamily which we call the superfamily.

The architecture of parallel β-helices and related folds

Three-dimensional structures have been determined of a large number of proteins characterized by a repetitive fold where each of the repeats (coils) supplies a strand to one or more parallel β-sheets. Some of these proteins form superfamilies of proteins, which have probably arisen by divergent evolution from a common ancestor. The classical example is the family including four families of pectinases without obviously related primary sequences, the phage P22 tailspike endorhamnosidase, chrondroitinase B and possibly pertactin from Bordetella pertusis. These show extensive stacking of similar residues to give aliphatic, aromatic and polar stacks such as the asparagine ladder. This suggests that coils can be added or removed by duplication or deletion of the DNA corresponding to one or more coils and explains how homologous proteins can have different numbers of coils. This process can also account for the evolution of other families of proteins such as the β-rolls, the leucine-rich repeat proteins, the hexapeptide repeat family, two separate families of β-helical antifreeze proteins and the spiral folds. These families need not be related to each other but will share features such as relative untwisted β-sheets, stacking of similar residues and turns between β-strands of approximately 90°often stabilized by hydrogen bonding along the direction of the parallel β-helix. Repetitive folds present special problems in the comparison of structures but offer attractive targets for structure prediction. The stacking of similar residues on a flat parallel β-sheet may account for the formation of amyloid with β-strands at right-angles to the fibril axis from many unrelated peptides.

Crystal Structure of Polygalacturonase from Erwinia carotovora ssp. carotovora

The crystal structure of the 40-kDa endo-polygalacturonase from Erwinia carotovora ssp.carotovora was solved by multiple isomorphous replacement and refined at 1.9 Å to a conventional crystallographicR-factor of 0.198 and R free of 0.239. This is the first structure of a polygalacturonase and comprises a 10 turn right-handed parallel β-helix domain with two loop regions forming a “tunnel like” substrate-binding cleft. Sequence conservation indicates that the active site of polygalacturonase is between these two loop regions, and comparison of the structure of polygalacturonase with that of rhamnogalacturonase A fromAspergillus aculeatus enables two conserved aspartates, presumed to be catalytic residues, to be identified. An adjacent histidine, in accord with biochemical results, is also seen. A similarity in overall electrostatic properties of the substrate-binding clefts of polygalacturonase and pectate lyase, which bind and cleave the same substrate, polygalacturonic acid, is also revealed.

Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases.

BACKGROUND Microbial pectin and pectate lyases are virulence factors that degrade the pectic components of the plant cell wall. The homogalacturan backbone of pectin varies in its degree of methylation from the highly methylated and relatively hydrophobic form known as pectin, to the fully demethylated and highly charged form known as pectate. Methylated and demethylated regions of pectin are cleaved by pectin lyase and calcium-dependent pectate lyases, respectively. Protein engineering of lyases specific for particular patterns of methylation, will yield modified pectins of high value to the food and pharmaceutical industries. RESULTS The crystal structures of pectin lyase A from two strains of Aspergillus niger, N400 and 4M-147, have been determined at pH 6.5 (2.4 A resolution) and pH 8.5 (1.93 A resolution), respectively. The structures were determined by a combination of molecular replacement, multiple isomorphous replacement and intercrystal averaging. Pectin lyase A folds into a parallel beta helix and shares many of the structural features of pectate lyases, despite no more than 17% sequence identity after pairwise structure-based alignment. These shared structural features include amino acid stacks and the asparagine ladder. However, the differences in the substrate-binding clefts of these two enzymes are striking. In pectin lyase A, the cleft is dominated by aromatic residues and is enveloped by negative electrostatic potential. In pectate lyases, this cleft is rich in charged residues and contains an elongated ribbon of positive potential when Ca2+ is bound. The major difference between the two pectin lyase A structures from the two strains is in the conformation of the loop formed by residues 182-187. These observed differences are due to the different pH values of crystallization. CONCLUSIONS The substrate-binding clefts and catalytic machinery of pectin and pectate lyases have diverged significantly. Specificity is dictated by both the nature of the protein-carbohydrate interaction and long-range electrostatic forces. Three potential catalytic residues have been identified in pectin lyase, two of these are common to pectate lyases. Pectin lyase A does not bind Ca2+ but an arginine residue is found in an equivalent position to the Ca2+ ion in pectate lyase, suggesting a similar role in catalysis. The activity of pectin lyase A is pH -dependent with an optimum activity at pH 5.5. The activity drops above pH 7.0 due to a conformational change at the binding cleft, triggered by the proximity of two buried aspartate residues.

Crystal structure of auxin-binding protein 1 in complex with auxin

The structure of auxin‐binding protein 1 (ABP1) from maize has been determined at 1.9 Å resolution, revealing its auxin‐binding site. The structure confirms that ABP1 belongs to the ancient and functionally diverse germin/seed storage 7S protein superfamily. The binding pocket of ABP1 is predominantly hydrophobic with a metal ion deep inside the pocket coordinated by three histidines and a glutamate. Auxin binds within this pocket, with its carboxylate binding the zinc and its aromatic ring binding hydrophobic residues including Trp151. There is a single disulfide between Cys2 and Cys155. No conformational rearrangement of ABP1 was observed when auxin bound to the protein in the crystal, but examination of the structure reveals a possible mechanism of signal transduction.

Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites.

BACKGROUND Sequence alignment suggests that xylanases evolved from two ancestral proteins and therefore can be grouped into two families, designated F and G. Family F enzymes show no sequence similarity with any known structure and their architecture is unknown. Studies of an inactive enzyme-substrate complex will help to elucidate the structural basis of binding and catalysis in the family F xylanases. RESULTS We have therefore determined the crystal structure of the catalytic domain of a family F enzyme, Pseudomonas fluorescens subsp. cellulosa xylanase A, at 2.5 A resolution and a crystallographic R-factor of 0.20. The structure was solved using an engineered catalytic core in which the nucleophilic glutamate was replaced by a cysteine. As expected, this yielded both high-quality mercurial derivatives and an inactive enzyme which enabled the preparation of the inactive enzyme-substrate complex in the crystal. We show that family F xylanases are eight-fold alpha/beta-barrels (TIM barrels) with two active-site glutamates, one of which is the nucleophile and the other the acid-base. Xylopentaose binds to five subsites A-E with the cleaved bond between subsites D and E. Ca2+ binding, remote from the active-site glutamates, stabilizes the structure and may be involved in the binding of extended substrates. CONCLUSIONS The architecture of P. fluorescens subsp. cellulosa has been determined crystallographically to be a commonly occurring enzyme fold, the eight-fold alpha/beta-barrel. Xylopentaose binds across the carboxy-terminal end of the alpha/beta-barrel in an active-site cleft which contains the two catalytic glutamates.

The prosequence of procaricain forms an α-helical domain that prevents access to the substrate-binding cleft

BACKGROUND Cysteine proteases are involved in a variety of cellular processes including cartilage degradation in arthritis, the progression of Alzheimers disease and cancer invasion: these enzymes are therefore of immense biological importance. Caricain is the most basic of the cysteine proteases found in the latex of Carica papaya. It is a member of the papain superfamily and is homologous to other plant and animal cysteine proteases. Caricain is naturally expressed as an inactive zymogen called procaricain. The inactive form of the protease contains an inhibitory proregion which consists of an additional 106 N-terminal amino acids; the proregion is removed upon activation. RESULTS The crystal structure of procaricain has been refined to 3.2 A resolution; the final model consists of three non-crystallographically related molecules. The proregion of caricain forms a separate globular domain which binds to the C-terminal domain of mature caricain. The proregion also contains an extended polypeptide chain which runs through the substrate-binding cleft, in the opposite direction to that of the substrate, and connects to the N terminus of the mature region. The mature region does not undergo any conformational change on activation. CONCLUSIONS We conclude that the rate-limiting step in the in vitro activation of procaricain is the dissociation of the prodomain, which is then followed by proteolytic cleavage of the extended polypeptide chain of the proregion. The prodomain provides a stable scaffold which may facilitate the folding of the C-terminal lobe of procaricain.

High resolution structure and sequence of T. aurantiacus Xylanase I: Implications for the evolution of thermostability in family 10 xylanases and enzymes with βα‐barrel architecture

Xylanase I is a thermostable xylanase from the fungus Thermoascus aurantiacus, which belongs to family 10 in the current classification of glycosyl hydrolases. We have determined the three‐dimensional X‐ray structure of this enzyme to near atomic resolution (1.14 Å) by molecular replacement, and thereby corrected the chemically determined sequence previously published. Among the five members of family 10 enzymes for which the structure has been determined, Xylanase I from T. aurantiacus and Xylanase Z from C. thermocellum are from thermophilic organisms. A comparison with the three other available structures of the family 10 xylanases from mesophilic organisms suggests that thermostability is effected mainly by improvement of the hydrophobic packing, favorable interactions of charged side chains with the helix dipoles and introduction of prolines at the N‐terminus of helices. In contrast to other classes of proteins, there is very little evidence for a contribution of salt bridges to thermostability in the family 10 xylanases from thermophiles. Further analysis of the structures of other proteins from thermophiles with eight‐fold βα‐barrel architecture suggests that favorable interactions of charged side chains with the helix dipoles may be a common way in which thermophilic proteins with this fold are stabilized. As this is the most common type of protein architecture, this finding may provide a useful guide for site‐directed mutagenesis aimed to improve the thermostability of βα‐barrel proteins. Proteins 1999;36:295–306.

Molecular basis of the activity of the phytopathogen pectin methylesterase

We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic‐esterase possessing parallel β‐helix architecture and now show that the two conserved aspartates are the nucleophile and general acid‐base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs.