Anaerobic synthesis of vitamin B12: characterization of the early steps in the pathway.

The anaerobic biosynthesis of vitamin B12 is slowly being unravelled. Recent work has shown that the first committed step along the anaerobic route involves the sirohydrochlorin (chelation of cobalt into factor II). The following enzyme in the pathway, CbiL, methylates cobalt-factor II to give cobalt-factor III. Recent progress on the molecular characterization of this enzyme has given a greater insight into its mode of action and specificity. Structural studies are being used to provide insights into how aspects of this highly complex biosynthetic pathway may have evolved. Between cobalt-factor III and cobyrinic acid, only one further intermediate has been identified. A combination of molecular genetics, recombinant DNA technology and bioorganic chemistry has led to some recent advances in assigning functions to the enzymes of the anaerobic pathway.

Substrate specificity and subsite mobility in T. aurantiacus xylanase 10A.

The substrate specificity of Thermoascus aurantiacus xylanase 10A (TAX) has been investigated both biochemically and structurally. High resolution crystallographic analyses at 291 K and 100 K of TAX complexes with xylobiose show that the ligand is in its alpha anomeric conformation and provide a rationale for specificity on p-nitrophenyl glycosides at the -1 and -2 subsites. Trp 275, which is disordered in uncomplexed structures, is stabilised by its interaction with xylobiose. Two structural subsets in family 10 are identified, which differ by the presence or absence of a short helical stretch in the eighth betaalpha-loop of the TIM barrel, the loop bearing Trp 275. This structural difference is discussed in the context of Trp 275 mobility and xylanase function.

Crystal structure of mannanase 26A from Pseudomonas cellulosa and analysis of residues involved in substrate binding.

The crystal structure of Pseudomonas cellulosa mannanase 26A has been solved by multiple isomorphous replacement and refined at 1.85 A resolution to an R-factor of 0.182 (R-free = 0.211). The enzyme comprises (beta/alpha)(8)-barrel architecture with two catalytic glutamates at the ends of beta-strands 4 and 7 in precisely the same location as the corresponding glutamates in other 4/7-superfamily glycoside hydrolase enzymes (clan GH-A glycoside hydrolases). The family 26 glycoside hydrolases are therefore members of clan GH-A. Functional analyses of mannanase 26A, informed by the crystal structure of the enzyme, provided important insights into the role of residues close to the catalytic glutamates. These data showed that Trp-360 played a critical role in binding substrate at the -1 subsite, whereas Tyr-285 was important to the function of the nucleophile catalyst. His-211 in mannanase 26A does not have the same function as the equivalent asparagine in the other GH-A enzymes. The data also suggest that Trp-217 and Trp-162 are important for the activity of mannanase 26A against mannooligosaccharides but are less important for activity against polysaccharides.

Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site.

Most structures of neutral lipases and esterases have been found to adopt the common alpha/beta hydrolase fold and contain a catalytic Ser-His-Asp triad. Some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom. Here, we report the crystal structure of pectin methylesterase that has neither the common alpha/beta hydrolase fold nor the common catalytic triad. The structure of the Erwinia chrysanthemi enzyme was solved by multiple isomorphous replacement and refined at 2.4 A to a conventional crystallographic R-factor of 17.9 % (R(free) 21.1 %). This is the first structure of a pectin methylesterase and reveals the enzyme to comprise a right-handed parallel beta-helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the alpha/beta hydrolase fold of rhamnogalacturonan acetylesterase with which it shares esterase activity. Pectin methylesterase has no significant sequence similarity with any protein of known structure. Sequence conservation among the pectin methylesterases has been mapped onto the structure and reveals that the active site comprises two aspartate residues and an arginine residue. These proposed catalytic residues, located on the solvent-accessible surface of the parallel beta-helix and in a cleft formed by external loops, are at a location similar to that of the active site and substrate-binding cleft of pectate lyase. The structure of pectin methylesterase is an example of a new family of esterases.

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 A resolution, comprises beta-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.

X-ray crystallographic study of xylopentaose binding to Pseudomonas fluorescens xylanase A.

The structure of the complex between a catalytically compromised family 10 xylanase and a xylopentaose substrate has been determined by X-ray crystallography and refined to 3.2 A resolution. The substrate binds at the C-terminal end of the eightfold betaalpha-barrel of Pseudomonas fluorescens subsp. cellulosa xylanase A and occupies substrate binding subsites -1 to +4. Crystal contacts are shown to prevent the expected mode of binding from subsite -2 to +3, because of steric hindrance to subsite -2. The loss of accessible surface at individual subsites on binding of xylopentaose parallels well previously reported experimental measurements of individual subsites binding energies, decreasing going from subsite +2 to +4. Nine conserved residues contribute to subsite -1, including three tryptophan residues forming an aromatic cage around the xylosyl residue at this subsite. One of these, Trp 313, is the single residue contributing most lost accessible surface to subsite -1, and goes from a highly mobile to a well-defined conformation on binding of the substrate. A comparison of xylanase A with C. fimi CEX around the +1 subsite suggests that a flatter and less polar surface is responsible for the better catalytic properties of CEX on aryl substrates. The view of catalysis that emerges from combining this with previously published work is the following: (1) xylan is recognized and bound by the xylanase as a left-handed threefold helix; (2) the xylosyl residue at subsite -1 is distorted and pulled down toward the catalytic residues, and the glycosidic bond is strained and broken to form the enzyme-substrate covalent intermediate; (3) the intermediate is attacked by an activated water molecule, following the classic retaining glycosyl hydrolase mechanism.

High resolution structure and sequence of T. aurantiacus xylanase I: implications for the evolution of thermostability in family 10 xylanases and enzymes with (beta)alpha-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 A) 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 (beta)alpha-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 (beta)alpha-barrel proteins. Proteins 1999;36:295-306.

Crystallization and preliminary X-ray analysis of arabinanase A from Pseudomonas fluorescens subspecies cellulosa.

Crystals of 1,5-alpha-arabinanase A from Pseudomonas fluorescens subspecies cellulosa have been obtained by vapour diffusion. The crystals belong to the space group P6122 with unit-cell parameters a = b = 91.6, c = 179.4 A with one molecule in the asymmetric unit. The native crystals and, to a much greater extent, heavy-atom soaked crystals are sensitive to radiation which necessitates cryocooling. Suitable cryocooling conditions have been established, though a shrinkage of the unit cell is observed, with a = b = 88.8 and c = 176.9 A.

Barley oxalate oxidase is a hexameric protein related to seed storage proteins: evidence from X-ray crystallography.

The oxalate oxidase enzyme expressed in barley roots is a thermostable, protease-resistant enzyme that generates H2O2. It has great medical importance because of its use to assay plasma and urinary oxalate, and it has also been used to generate transgenic, pathogen-resistant crops. This protein has now been purified and three types of crystals grown. X-ray analysis shows that the symmetry present in these crystals is consistent with a hexameric arrangement of subunits, probably a trimer of dimers. This structure may be similar to that found in the related seed storage proteins.

Crystallization and preliminary X-ray diffraction studies of a family 26 endo-beta-1,4 mannanase (ManA) from Pseudomonas fluorescens subspecies cellulosa.

Crystals of an endo-beta-1,4-mannanase (1,4-beta-D-mannohydrolase, E. C. 3.2.1.78) from Pseudomonas fluorescens sub species cellulosa have been grown by the hanging-drop technique at 291 K over a period of one to two weeks to maximal dimensions of 0.17 x 0.17 x 0.25 mm. These crystals belong to the space group R32 (or R3) with cell dimensions of a = b = 155.4 and c = 250.8 A (hexagonal setting) and contain three (six) molecules in the asymmetric unit. The crystals diffract to at least 3.2 A using a laboratory source and are suitable for structure determination.

Structural basis of the properties of an industrially relevant thermophilic xylanase.

A thermophilic xylanase from Bacillus strain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized. The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry. The xylanase is stable at 60 degrees C and relatively stable at high temperatures, with a temperature optimum of 75 degrees C. The pH optimum is 6, but the enzyme is active over a broad pH range. The xylanase has been cloned and sequenced, and the crystal structure has been determined. The structure of Bacillus D3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or "sticky patches" between pairs of molecules. These "sticky patches" on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol. The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase.

Calcium protects a mesophilic xylanase from proteinase inactivation and thermal unfolding.

Crystal structure analysis of Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA) indicated that the enzyme contained a single calcium binding site that did not exhibit structural features typical of the EF-hand motif. Isothermal titration calorimetry revealed that XYLA binds calcium with a Ka of 4.9 x 10(4) M-1 and a stoichiometry consistent with one calcium binding site per molecule of enzyme. Occupancy of the calcium binding domain with its ligand protected XYLA from proteinase and thermal inactivation and increased the melting temperature of the enzyme from 60.8 to 66.5 degrees C. However, the addition of calcium or EDTA did not influence the catalytic activity of the xylanase. Replacement of the calcium binding domain, which is located within loop 7 of XYLA, with the corresponding short loop from Cex (a Cellulomonas fimi xylanase/exoglucanase), did not significantly alter the biochemical properties of the enzyme. These data suggest that the primary function of the calcium binding domain is to increase the stability of the enzyme against thermal unfolding and proteolytic attack. To understand further the nature of the calcium binding domain of XYLA, four variants of the xylanase, D256A, N261A, D262A, and XYLA"', in which Asp-256, Asn-261, and Asp-262 had all been changed to alanine, were constructed. These mutated enzymes did not show any significant binding to Ca2+, indicating that Asp-256, Asn-261, and Asp-262 play a pivotal role in the affinity of XYLA for the divalent cation. In the presence or absence of calcium, XYLA"' exhibited thermal stability similar to that of the native enzyme bound to Ca2+ ions, although the variant was sensitive to proteinase inactivation. The role of the calcium binding domain in vivo and the possible mechanism by which the domain evolved are discussed.

Crystallization and preliminary X-ray analysis of the major endoglucanase from Thermoascus aurantiacus.

The major endoglucanase (35 kDa) from the thermophilic fungus Thermoascus aurantiacus has been purified from culture filtrates using an affinity method and the sequence for 35 N-terminal amino acids determined. This has allowed assignment of the enzyme to subtype A6 of family 5 endoglucanases. The enzyme has been crystallized as thick plates by the hanging-drop method using ammonium sulfate as precipitant. The crystals belong to space group P2(1)2(1)2(1) with cell edges a = 76.4, b = 85.7 and c = 89.5 A, with two molecules in the asymmetric unit, and diffract to 1.62 A resolution using synchrotron radiation. The structure will be solved by isomorphous replacement.

The prosequence of procaricain forms an alpha-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 Alzheimer's 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.

Crystal structure of a caricain D158E mutant in complex with E-64.

The structure of the D158E mutant of caricain (previously known as papaya protease omega) in complex with E-64 has been determined at 2.0 A resolution (overall R factor 19.3%). The structure reveals that the substituted glutamate makes the same pattern of hydrogen bonds as the aspartate in native caricain. This was not anticipated since in the native structure there is insufficient room to accommodate the glutamate side chain. The glutamate is accommodated in the mutant by a local expansion of the structure demonstrating that small structural changes are responsible for the change in activity.

Refined crystal structure of the catalytic domain of xylanase A from Pseudomonas fluorescens at 1.8 A resolution.

The three-dimensional structure of native xylanase A from Pseudomonas flouorescens subspecies cellulosa has been refined at 1.8 A resolution. The space group is P2(1)2(1)2(1) with four molecules in the asymmetric unit. The final model has an R factor of 0.166 for 103 749 reflections with the four molecules refined independently. The tertiary structure consists of an eightfold beta/alpha-barrel, the so-called TIM-barrel fold. The active site is in an open cleft at the carboxy-terminal end of the beta/alpha-barrel, and the active-site residues are a pair of glutamates, Glu127 on strand 4 and Glu246 on strand 7. Both these catalytic glutamate residues are found on beta-bulges. An atypically long loop after strand 7 is stabilized by calcium. Unusual features include a non-proline cis-peptide residue Ala80 which is found on a beta-bulge at the end of beta-strand 3. The three beta-bulge type distortions occurring on beta-strands 3, 4 and 7 are functionally significant as they serve to orient important active-site residues. The active-site residues are further held in place by an extensive hydrogen-bonding network of active-site residues in the catalytic site of xylanase A. A chain of well ordered water molecules occupies the substrate-binding cleft, some or all of which are expelled on binding of the substrate.

The potent mitogen Pasteurella multocida toxin is highly resistant to proteolysis but becomes susceptible at lysosomal pH.

The susceptibility of the potent mitogen Pasteurella multocida toxin (PMT) to various proteases was investigated. PMT at a toxin to protease molar ratio of 1:1 was resistant to 8 of the 11 proteases tested after one hour. With longer incubation, PMT remained resistant to 7 proteases, and this correlated with a retention of biological activity, indicating that PMT might not require proteolytic cleavage at least until it bound to a cell receptor. Previous evidence had suggested that PMT is processed in the cell via an endosome or lysosome. We have shown that PMT became susceptible to proteolysis when the pH was lowered to 5 or below. This supports the previous suggestion that PMT is processed via a low pH compartment in the cell.

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.

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions.

The role of electrostatic interactions between the ionizable Asp158 and the active site thiolate-imidazolium ion pair of some cysteine proteinases has been the subject of controversy for some time. This study reports the expression of wild type procaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants from Escherichia coli. Purification of autocatalytically matured enzymes yielded sufficient fully active material for pH (kcat/Km) profiles to be obtained. Use of both uncharged and charged substrates allowed the effects of different reactive enzyme species to be separated from the complications of electrostatic effects between enzyme and substrate. At least three ionizations are detectable in the acid limb of wild type caricain and the Glu and Asn mutants. Only two pKa values, however, are detectable in the acid limb using the Ala mutant. Comparison of pH activity profiles shows that whilst an ionizable residue at position 158 is not essential for the formation of the thiolate-imidazolium ion pair, it does form a substantial part of the electrostatic field responsible for increased catalytic competence. Changing the position of this ionizable group in any way reduces activity. Complete removal of the charged group reduces catalytic competence even further. This work indicates that hydronations distant to the active site are contributing to the electrostatic effects leading to multiple active ionization states of the enzyme.

Modification of the head-group selectivity of porcine pancreatic phospholipase A2 by protein engineering.

On the basis of the three-dimensional structures of phospholipid and porcine pancreatic phospholipase A2 (pla2), it was predicted that the removal of a negative charge in the hydrophilic region of the phospholipid binding site would influence the head-group selectivity of porcine pancreatic pla2. To test this prediction, glutamic acid 46 was changed to leucine by site-directed mutagenesis. The E46L mutant, expressed in Escherichia coli, was purified and characterized. The mutation did not affect the activity toward the mixed micellar substrate, but the activity of E46L toward DiC12-P, which has two negative charges on the head group, was three times higher than that of DiC12-PC, which carries no net charge in the head group. The native pla2 was inhibited by the product(s) released from DiC12-P but not the mutant enzyme. Kinetic analysis revealed that the E46L mutant and the native pla2 had comparable affinities (Km) toward monomeric and micellar phospholipids of zwitterionic type while the activity (kcat) of E46L, toward the same substrates, was approximately 50% lower compared to that of native pla2. When micellar DiC12-P was used as a substrate, the Kmapp value for E46L was four times lower and the kcatapp/kmapp was 5-fold higher than those of native pla2. However, the kinetic parameters of mutant and native pla2s remained unchanged for monomeric HEPG, with one negative charge in the head group. Thus, we have modified the head-group selectivity of porcine pancreatic pla2 by protein engineering.