Improving the 'tool box' for robust industrial enzymes.

The speed of sequencing of microbial genomes and metagenomes is providing an ever increasing resource for the identification of new robust biocatalysts with industrial applications for many different aspects of industrial biotechnology. Using 'natures catalysts' provides a sustainable approach to chemical synthesis of fine chemicals, general chemicals such as surfactants and new consumer-based materials such as biodegradable plastics. This provides a sustainable and 'green chemistry' route to chemical synthesis which generates no toxic waste and is environmentally friendly. In addition, enzymes can play important roles in other applications such as carbon dioxide capture, breakdown of food and other waste streams to provide a route to the concept of a 'circular economy' where nothing is wasted. The use of improved bioinformatic approaches and the development of new rapid enzyme activity screening methodology can provide an endless resource for new robust industrial biocatalysts.This mini-review will discuss several recent case studies where industrial enzymes of 'high priority' have been identified and characterised. It will highlight specific hydrolase enzymes and recent case studies which have been carried out within our group in Exeter.

The development and evaluation of a conducting matrix for the electrochemical regeneration of the immobilised co-factor NAD(H) under continuous flow.

Through the preparation of a novel controlled pore glass-poly(pyrrole) material we have developed a conducting support that is not only suitable for the co-immobilisation of enzymes and co-factors, but also enables the facile electrochemical regeneration of the co-factor during a reaction. Employing the selective reduction of (rac)-2-phenylpropionaldehyde to (S)-phenyl-1-propanol as a model, we have demonstrated the successful co-immobilisation of the HLADH enzyme and co-factor NAD(H); with incorporation of the material into a continuous flow reactor facilitating the in situ electrochemical regeneration of NAD(H) for in excess of 100 h. Using this approach we have developed a reagent-less, atom efficient system applicable to the cost-effective, continuous biosynthesis of chiral compounds.

A data-based reaction mechanism for type I fructose bisphosphate aldolase.

The structures of three type I fructose-1,6-bisphosphate aldolases have been determined and the common residues surrounding the Schiff base-forming Lys residue located. Armed with this information, it is now possible to propose a mechanism for this ubiquitous enzyme which is consistent with the recorded biochemical data. An interesting, but by no means mandatory, feature of the reaction mechanism is that catalysis can proceed without exchange with the solvent.

The atomic-resolution structure of a novel bacterial esterase.

BACKGROUND: A novel bacterial esterase that cleaves esters on halogenated cyclic compounds has been isolated from an Alcaligenes species. This esterase 713 is encoded by a 1062 base pair gene. The presence of a leader sequence of 27 amino acids suggests that this enzyme is exported from the cytosol. Esterase 713 has been over-expressed in Agrobacterium without this leader sequence. Its amino acid sequence shows no significant homology to any known protein sequence. RESULTS: The crystal structure of esterase 713 has been determined by multiple isomorphous replacement and refined to 1. 1 A resolution. The subunits of this dimeric enzyme comprise a single domain with an alpha/beta hydrolase fold. The catalytic triad has been identified as Ser206-His298-Glu230. The acidic residue of the catalytic triad (Glu230) is located on the beta6 strand of the alpha/beta hydrolase fold, whereas most other alpha/beta hydrolase enzymes have the acidic residue located on the beta7 strand. The oxyanion hole is formed by the mainchain nitrogens of Cys71 and Gln207 as identified by the binding of a substrate analogue, (S)-7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1, 4-benzodiazepine-2-acetic acid. Cys71 forms a disulphide bond with the neighbouring Cys72. CONCLUSIONS: Despite negligible sequence homology, esterase 713 has structural similarities to a number of other esterases and lipases. Residues of the oxyanion hole were confirmed by structural comparison with Rhizomucor miehei lipase. It is proposed that completion of a functional active site requires the formation of the disulphide bond between adjacent residues Cys71 and Cys72 on export of the esterase into the oxidising environment of the periplasmic space.

Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution.

BACKGROUND: The peroxiredoxins (Prxs) are an emerging family of multifunctional enzymes that exhibit peroxidase activity in vitro, and in vivo participate in a range of cellular processes known to be sensitive to reactive oxygen species. Thioredoxin peroxidase B (TPx-B), a 2-Cys type II Prx from erythrocytes, promotes potassium efflux and down-regulates apoptosis and the recruitment of monocytes by endothelial tissue. RESULTS: The crystal structure of human decameric TPx-B purified from erythrocytes has been determined to 1.7 [corrected)] A resolution. The structure is a toroid comprising five dimers linked end-on through predominantly hydrophobic interactions, and is proposed to represent an intermediate in the in vivo reaction cycle. In the crystal structure, Cys51, the site of peroxide reduction, is oxidised to cysteine sulphinic acid. The residue Cys172, lies approximately 10 A away from Cys51 [corrected]. CONCLUSIONS: The oxidation of Cys51 appears to have trapped the structure into a stable decamer, as confirmed by sedimentation analysis. A comparison with two previously reported dimeric Prx structures reveals that the catalytic cycle of 2-Cys Prx requires significant conformational changes that include the unwinding of the active-site helix and the movement of four loops. It is proposed that the stable decamer forms in vivo under conditions of oxidative stress. Similar decameric structures of TPx-B have been observed by electron microscopy, which show the protein associated with the erythrocyte membrane.

Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 A crystal structure of the glutaminase domain of glucosamine 6-phosphate synthase.

BACKGROUND: Amidotransferases use the amide nitrogen of glutamine in a number of important biosynthetic reactions. They are composed of a glutaminase domain, which catalyzes the hydrolysis of glutamine to glutamate and ammonia, and a synthetase domain, catalyzing amination of the substrate. To gain insight into the mechanism of nitrogen transfer, we examined the structure of the glutaminase domain of glucosamine 6-phosphate synthase (GLMS). RESULTS: The crystal structures of the enzyme complexed with glutamate and with a competitive inhibitor, Glu-hydroxamate, have been determined to 1.8 A resolution. The protein fold has structural homology to other members of the superfamily of N-terminal nucleophile (Ntn) hydrolases, being a sandwich of antiparallel beta sheets surrounded by two layers of alpha helices. CONCLUSIONS: The structural homology between the glutaminase domain of GLMS and that of PRPP amidotransferase (the only other Ntn amidotransferase whose structure is known) indicates that they may have diverged from a common ancestor. Cys1 is the catalytic nucleophile in GLMS, and the nucleophilic character of its thiol group appears to be increased through general base activation by its own alpha-amino group. Cys1 can adopt two conformations, one active and one inactive; glutamine binding locks the residue in a predetermined conformation. We propose that when a nitrogen acceptor is present Cys1 is kept in the active conformation, explaining the phenomenon of substrate-induced activation of the enzyme, and that Arg26 is central in this coupling.

The secondary structure of salt-extracted ribosomal proteins from Escherichia coli as studied by circular dichroic spectroscopy.

Ribosomal proteins from Escherichia coli MRE600 have been obtained by a new, mild purification procedure. This involves extraction of the subunits with salt followed by chromatographic fractionation in the presence of salt. The use of urea or other denaturing agents and conditions is avoided. A survey of the secondary structure of the 30 S and 50 S proteins, as observed by circular dichroic spectroscopy, is presented. The spectra have been analysed by a new procedure which uses a library of 16 circular dichroic spectra of proteins with a known three-dimensional structure. This method provides a more reliable analysis, especially of the contribution from beta-sheet. The results show that most of the 30 S proteins have a high alpha-helix content, whereas the 50 S proteins are more diverse. The latter group shows a larger contribution from beta-sheet. The data presented here are compared with those already published for a number of proteins which were, with one exception, prepared in the presence of urea. In most cases we find higher alpha-helix and beta-sheet values for the salt-extracted proteins than for the corresponding urea-treated proteins. In those cases, however, where special care was taken to renature the urea-treated proteins agreement is found to within the expected experimental error. The results show that salt-extracted ribosomal proteins have a well-defined secondary structure with a relatively small contribution from unordered structure.

Comparison of the decameric structure of peroxiredoxin-II by transmission electron microscopy and X-ray crystallography.

The decameric human erythrocyte protein torin is identical to the thiol-specific antioxidant protein-II (TSA-II), also termed peroxiredoxin-II (Prx-II). Single particle analysis from electron micrographs of Prx-II molecules homogeneously orientated across holes in the presence of a thin film of ammonium molybdate and trehalose has facilitated the production of a >/=20 A 3-D reconstruction by angular reconstitution that emphasises the D5 symmetry of the ring-like decamer. The X-ray structure for Prx-II was fitted into the transmission electron microscopic reconstruction by molecular replacement. The surface-rendered transmission electron microscopy (TEM) reconstruction correlates well with the solvent-excluded surface of the X-ray structure of the Prx-II molecule. This provides confirmation that transmission electron microscopy of negatively stained specimens, despite limited resolution, has the potential to reveal a valid representation of surface features of protein molecules. 2-D crystallisation of the Prx-II protein on mica as part of a TEM study resulted in the formation of a p2 crystal form with parallel linear arrays of stacked rings. This latter 2-D form correlates well with that observed from the 2.7 A X-ray structure of Prx-II solved from a new orthorhombic 3-D crystal form.

Purification, crystallization and preliminary X-ray analysis of the 3-phosphoglycerate kinase from Bacillus stearothermophilus.

As part of a programme investigating the molecular basis of thermal stability in proteins we have isolated and characterized the thermally stable 3-phosphoglycerate kinase (PGK) from Bacillus stearothermophilus NCA 1503. The B. stearothermophilus PGK has been crystallized in a form suitable for X-ray diffraction analysis. Crystals which diffract to greater than 1.8 A resolution have been grown in the presence of the nucleotide substrate, MgATP, using polyethylene glycol (PEG 600) as a precipitant. The best crystals have been obtained using "seeding" techniques and are monoclinic, space group P2(1), with cell dimensions a = 40.5 A, b = 74.0 A, c = 68.5 A and beta = 99.8 degrees.

The crystal structure of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus in the presence of NADP(+) at 2.1 A resolution.

The crystal structure of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaeon Methanothermus fervidus has been solved in the holo form at 2.1 A resolution by molecular replacement. Unlike bacterial and eukaryotic homologous enzymes which are strictly NAD(+)-dependent, GAPDH from this organism exhibits a dual-cofactor specificity, with a marked preference for NADP(+) over NAD(+). The present structure is the first archaeal GAPDH crystallized with NADP(+). GAPDH from M. fervidus adopts a homotetrameric quaternary structure which is topologically similar to that observed for its bacterial and eukaryotic counterparts. Within the cofactor-binding site, the positively charged side-chain of Lys33 decisively contributes to NADP(+) recognition through a tight electrostatic interaction with the adenosine 2'-phosphate group. Like other GAPDHs, GAPDH from archaeal sources binds the nicotinamide moiety of NADP(+) in a syn conformation with respect to the adjacent ribose and so belongs to the B-stereospecific class of oxidoreductases. Stabilization of the syn conformation is principally achieved through hydrogen bonding of the carboxamide group with the side-chain of Asp171, a structural feature clearly different from what is observed in all presently known GAPDHs from bacteria and eukaryotes. Within the catalytic site, the reported crystal structure definitively confirms the essential role previously assigned to Cys140 by site-directed mutagenesis studies. In conjunction with new mutation results reported in this paper, inspection of the crystal structure gives reliable evidence for the direct implication of the side-chain of His219 in the catalytic mechanism. M. fervidus grows optimally at 84 degrees C with a maximal growth temperature of 97 degrees C. The paper includes a detailed comparison of the present structure with four other homologous enzymes extracted from mesophilic as well as thermophilic organisms. Among the various phenomena related to protein thermostabilization, reinforcement of electrostatic and hydrophobic interactions as well as a more efficient molecular packing appear to be essentially promoted by the occurrence of two additional alpha-helices in the archaeal GAPDHs. The first one, named alpha4, is located in the catalytic domain and participates in the enzyme architecture at the quaternary structural level. The second one, named alphaJ, occurs at the C terminus and contributes to the molecular packing within each monomer by filling a peripherical pocket in the tetrameric assembly.

Activity and specificity of human aldolases.

The structure of the type I fructose 1,6-bisphosphate aldolase from human muscle has been extended from 3 A to 2 A resolution. The improvement in the resulting electron density map is such that the 20 or so C-terminal residues, known to be associated with activity and isozyme specificity, have been located. The side-chain of the Schiff's base-forming lysine 229 is located towards the centre of an eight-stranded beta-barrel type structure. The C-terminal "tail" extends from the rim of the beta-barrel towards lysine 229, thus forming part of the active site of the enzyme. This structural arrangement appears to explain the difference in activity and specificity of the three tissue-specific human aldolases and helps with our understanding of the type I aldolase reaction mechanism.

Crystal structure of dodecameric vanadium-dependent bromoperoxidase from the red algae Corallina officinalis.

The three-dimensional structure of the vanadium bromoperoxidase protein from the marine red macroalgae Corallina officinalis has been determined by single isomorphous replacement at 2.3 A resolution. The enzyme subunit is made up of 595 amino acid residues folded into a single alpha+beta domain. There are 12 bromoperoxidase subunits, arranged with 23-point group symmetry. A cavity is formed by the N terminus of each subunit in the centre of the dodecamer. The subunit fold and dimer organisation of the Cor. officinalis vanadium bromoperoxidase are similar to those of the dimeric enzyme from the brown algae Ascophyllum nodosum, with which it shares 33 % sequence identity. The different oligomeric state of the two algal enzymes seems to reflect separate mechanisms of adaptation to harsh environmental conditions and/or to chemically active substrates and products. The residues involved in the vanadate binding are conserved between the two algal bromoperoxidases and the vanadium chloroperoxidase from the fungus Curvularia inaequalis. However, most of the other residues forming the active-site cavity are different in the three enzymes, which reflects differences in the substrate specificity and stereoselectivity of the reaction. A dimer of the Cor. officinalis enzyme partially superimposes with the two-domain monomer of the fungal enzyme.

Crystal structure of the glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus.

The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaea shows low sequence identity (16-20%) with its eubacterial and eukaryotic counterparts. The crystal structure of the apo GAPDH from Sulfolobus solfataricus has been determined by multiple isomorphous replacement at 2.05 A resolution. The enzyme has several differences in secondary structure when compared with eubacterial GAPDHs, with an overall increase in the number of alpha-helices. There is a relocation of the active-site residues within the catalytic domain of the enzyme. The thermostability of the S. solfataricus enzyme can be attributed to a combination of an ion pair cluster and an intrasubunit disulphide bond.

Crystal structure of human muscle aldolase complexed with fructose 1,6-bisphosphate: mechanistic implications.

Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.

Sequence and expression of the gene encoding 3-phosphoglycerate kinase from Bacillus stearothermophilus.

The structural gene (pgk) encoding 3-phosphoglycerate (PGK) from Bacillus stearothermophilus NCA1503, has been cloned in Escherichia coli and its complete nucleotide sequence determined. The gene consists of an open reading frame corresponding to a protein of 394 amino acids (aa) (calculated Mr 42,703) and, in common with other prokaryotic pgk genes, is preceded by the structural gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Constructs containing the B. stearothermophilus pgk gene and its flanking sequences in the high-copy plasmid, pUC9, co-express both PGK and GAPDH at high levels in transformed E. coli cells, typically producing PGK at levels of up to 30% of the soluble cell protein. The deduced aa sequence of B. stearothermophilus PGK is compared with those of the mesophilic (yeast) and extreme thermophilic (Thermus thermophilus) enzymes since the crystal structure of these PGKs are known or are in the process of being determined. Changes in the sequences of the three enzymes, as they appear to relate to the enhancement of thermal stability, are discussed.

Cloning and sequencing of a gene from the archaeon Pyrococcus furiosus with high homology to a gene encoding phosphoenolpyruvate synthetase from Escherichia coli.

A gene from the hyperthermophilic archaeon Pyrococcus furiosus, strain Vc1 (DSM 3638), contains an 817-amino-acid open reading frame which shows 42% identity to the phosphoenolpyruvate (PEP) synthetase of Escherichia coli. This putative P. furiosus PEP synthetase is slightly larger than the E. coli enzyme, the region between residues 58 and 89 being absent from the latter.

Site-directed mutagenesis of yeast phosphoglycerate kinase. Arginines 65, 121 and 168.

In the absence of a structure of the closed form of phosphoglycerate kinase we have modified by site directed mutagenesis several of the residues which, on the basis of the open form structure, are likely to be involved in substrate binding and catalysis. Here we report on the kinetic and anion activation properties of the yeast enzyme modified at positions 65, 121 and 168. In each case an arginine, thought to be involved in the binding of the sugar substrate's non-transferable phosphate group, has been replaced by lysine (same charge) and by methionine (no charge). Km values for 3-phosphoglycerate of all six mutant enzymes are only marginally higher than that of the wild-type enzyme. Removing the charge associated with two of the three arginine residues appears to influence (as judged by the measured Km's) the binding of ATP. Although binding affinity is not necessarily coupled to turnover the substitutions which have the greatest effect on the Km's do correlate with the reduction in enzymes maximum velocity. The one exception to this generalisation is the R65K mutant which, surprisingly, has a significantly higher kcat than the wild-type enzyme. In the open form structure of the pig muscle enzyme each of the three substituted arginines residues are seen to make two hydrogen bonds to the sugar substrate's non-transferable phosphate. From this it might be expected that anion activation would be similarly affected by the substitution of any one of these three residues. Although the interpretation of such effects are complicated by the fact that one of the mutants (R65M) unfolds at low salt concentrations, this appears not to be the case. Replacing Arg121 and Arg168 with methionine reduces the anion activation whereas a lysine in either of these two positions practically destroys the effect. With the substitutions at residue 65 the opposite is observed in that the lysine mutant shows anion activation whereas the methionine mutant does not.

The crystal structure of human muscle aldolase at 3.0 A resolution.

The three-dimensional structure of fructose-1,6-bisphosphate aldolase from human muscle has been determined at 3.0 A resolution by X-ray crystallography. The active protein is a tetramer of 4 identical subunits each of which is composed of an eight-stranded alpha/beta-barrel structure. The lysine residue responsible for Schiff base formation with the substrate is located near the centre of the barrel in the middle of the sixth beta-strand. While the overall topology of the alpha/beta-barrel is very similar to those found in several other enzymes, the distribution of charged residues inside the core of the barrel seems distinct. The quaternary fold of human muscle aldolase uses interfacial regions also involved in the subunit association of other alpha/beta-barrel proteins found in glycolysis, but exploits these regions in a manner not seen previously.

Site-directed mutagenesis of histidine 62 in the 'basic patch' region of yeast phosphoglycerate kinase.

Site-directed mutagenesis has been used to produce a mutant form of yeast phosphoglycerate kinase (PGK) in which the 'basic patch' residue His 62 has been replaced by a glutamine residue. Using 1H-NMR spectroscopy, it was found that 3-phosphoglycerate (3-PG) binding to the mutant protein induces the same conformational effects as for wild-type PGK, although the affinity was reduced by 2- to 3-fold. Kinetic studies show both Km for 3-PG and Vmax to be increased by approximately 2-fold relative to the wild-type enzyme. These data are consistent with the suggestion that His 62 assists in the binding of the substrate to the enzyme.

Studies with inhibitors of the glycolytic enzyme phosphoglycerate kinase for potential treatment of cardiovascular and respiratory disorders.

Inhibition of the glycolytic enzyme phosphoglycerate kinase (PGK) in erythrocyte cells could provide a method of treatment for cardiovascular and respiratory disorders. The product of the reaction catalysed by PGK, 1,3-diphosphoglycerate, is converted by another enzyme in erythrocytes to 2,3-diphosphoglycerate, which is an allosteric effector of haemoglobin. For this reason, a series of fluoro-phosphonate inhibitors have been tested for their potency in detailed inhibition kinetic experiments with yeast PGK. The results were analysed by Lineweaver-Burk and Dixon plots and Ki values obtained. Two fluorophosphonates were found to be inhibitory and both have an electron rich mid-chain functionality, which is thought to provide electrons for hydrogen bonding to residues in the triose binding site of the enzyme. It is postulated that either the fluorine or mid-chain moieties of the analogues are binding to Asp23 and Asn25 residues in the so called 'basic patch' area of the triose site. These residues are shown to bind to the D-hydroxyl moiety on the C2 of the true substrate, 3-phosphoglycerate, in the high-resolution crystal structure of pig muscle PGK co-crystallized with 3-phosphoglycerate.