Molecular dynamics investigations of BioH protein substrate specificity for biotin synthesis

BioH, an enzyme of biotin synthesis, plays an important role in fatty acid synthesis which assembles the pimelate moiety. Pimeloyl-acyl carrier protein (ACP) methyl ester, which is long known to be a biotin precursor, is the physiological substrate of BioH. Azelayl methyl ester, which has a longer chain than pimeloyl methyl ester, conjugated to ACP is also indeed accepted by BioH with very low rate of hydrolysis. To date, the substrate specificity for BioH and the molecular origin for the experimentally observed rate changes of hydrolysis by the chain elongation have remained elusive. To this end, we have investigated chain elongation effects on the structures by using the fully atomistic molecular dynamics simulations combined with binding free energy calculations. The results indicate that the substrate specificity is determined by BioH together with ACP. The added two methylenes would increase the structural flexibility by protein motions at the interface of ACP and BioH, instead of making steric clashes with the side chains of the BioH hydrophobic cavity. On the other hand, the slower hydrolysis of azelayl substrate is suggested to be associated with the loose of contacts between BioH and ACP, and with the lost electrostatic interactions of two ionic/hydrogen bonding networks at the interface of the two proteins. The present study provides important insights into the structure–function relationships of the complex of BioH with pimeloyl-ACP methyl ester, which could contribute to further understanding about the mechanism of the biotin synthetic pathway, including the catalytic role of BioH.     

The Serratia marcescens bioH gene encodes an esterase

The 3.9 kb chromosomal DNA was cloned from Serratia marcescens Sr41, which confers on Escherichia coli cells a phenotype of clear halo formation on tributyrin agar plates. Three complete open reading frames (ORFs) were identified in the inserted DNA, and one ORF was demonstrated to encode a 28 kDa protein of 255 amino acids related to esterase activity. Interestingly, the ORF was 70% identical to a product of the E. coli bioH gene, which lies at a locus separated from the bioABFCD operon and acts in the early steps of the biotin synthetic pathway before pimeloyl-CoA synthesis. This gene complemented a bioH-deficient mutation of E. coli. From the sequence analysis, BioH is presumed to be a serine hydrolase, which belongs to the alpha/beta hydrolase-fold family comprising a wide variety of hydrolases including esterases. A catalytic triad composed of a nucleophilic residue (Ser80), an acidic residue (Asp206), and histidine (His234) was conserved in BioH, and the nucleophilic residue Ser, a catalytic center, was situated in the consensus sequence of G-X-S-X-G-G, a nucleophile elbow. Although the enzymatic function of BioH is not yet elucidated, the bioH gene products from S. marcescens and E. coli show esterase activity, which may imply the hydrolysis of a precursor leading to pimeloyl-CoA ester. The esterase activity of BioH and its CoA binding activity recently reported agree with a current hypothesis of pimeloyl-CoA ester synthesis from CoA and acylester derivatives including an acyl-carrier protein.     

A structural model of the plant acyl-acyl carrier protein thioesterase FatB comprises two helix/4-stranded sheet domains, the N-terminal domain containing residues that affect specificity and the C-terminal domain containing catalytic residues

Plant acyl-acyl carrier protein thioesterases (TEs) terminate the acyl-acyl carrier protein track of fatty acid biosynthesis and play an essential role in determining the amount and composition of fatty acids entering the storage lipid pool. A combination of bioinformatics tools was used to predict a three-dimensional model for Arabidopsis FatB (AtFatB), which comprises a fold similar to that of Escherichia coli TEII, an enzyme that is functionally similar to plant TEs but lacks significant sequence similarity and displays different inhibitor sensitivity. The catalytic residues in AtFatB, Cys-264 and His-229, localize to the same region of the model as catalytic residues found in other enzymes with helix/multi-stranded sheet motifs (hot dog folds). Based on the model, we identified Asn-227 as a possible third member of the proposed papain-like catalytic triad. The conversion of Asn-227 to Ala resulted in a loss of detectable activity (>200-fold reduction), similar to the result seen for the equivalent mutation in papain. Mapping of plant TE specificity-affecting mutations onto the structural model showed that these mutations all cluster around the catalytic triad. Also, superposition of the crystallographically determined structures of the complexes of 4-hydroxybenzoyl-CoA TE with substrate and beta-hydroxydecanoyl thiol ester dehydrase with inhibitor onto the AtFatB model showed that the substrate and inhibitor localize to the same region as the AtFatB catalytic triad in their respective structures. Together these data corroborate the structural model and show that the hot dog fold is common to enzymes from both prokaryotes and eukaryotes and that this fold supports at least three different catalytic mechanisms.     

Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity

Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.     

Three enigmatic BioH isoenzymes are programmed in the early stage of mycobacterial biotin synthesis,an attractive anti-TB drug target

Tuberculosis (TB) is one of the leading infectious diseases of global concern, and one quarter of the world’s population are TB carriers. Biotin metabolism appears to be an attractive anti-TB drug target. However, the first-stage of mycobacterial biotin synthesis is fragmentarily understood. Here we report that three evolutionarily-distinct BioH isoenzymes (BioH1 to BioH3) are programmed in biotin synthesis of Mycobacterium smegmatis. Expression of an individual bioH isoform is sufficient to allow the growth of an Escherichia coli ΔbioH mutant on the non-permissive condition lacking biotin. The enzymatic activity in vitro combined with biotin bioassay in vivo reveals that BioH2 and BioH3 are capable of removing methyl moiety from pimeloyl-ACP methyl ester to give pimeloyl-ACP, a cognate precursor for biotin synthesis. In particular, we determine the crystal structure of dimeric BioH3 at 2.27Å, featuring a unique lid domain. Apart from its catalytic triad, we also dissect the substrate recognition of BioH3 by pimeloyl-ACP methyl ester. The removal of triple bioH isoforms (ΔbioH1/2/3) renders M. smegmatis biotin auxotrophic. Along with the newly-identified Tam/BioC, the discovery of three unusual BioH isoforms defines an atypical ‘BioC-BioH(3)’ paradigm for the first-stage of mycobacterial biotin synthesis. This study solves a long-standing puzzle in mycobacterial nutritional immunity, providing an alternative anti-TB drug target.     

Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase

Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.     

Protein surface analysis for function annotation in high-throughput structural genomics pipeline

Structural genomics (SG) initiatives are expanding the universe of protein fold space by rapidly determining structures of proteins that were intentionally selected on the basis of low sequence similarity to proteins of known structure. Often these proteins have no associated biochemical or cellular functions. The SG success has resulted in an accelerated deposition of novel structures. In some cases the structural bioinformatics analysis applied to these novel structures has provided specific functional assignment. However, this approach has also uncovered limitations in the functional analysis of uncharacterized proteins using traditional sequence and backbone structure methodologies. A novel method, named pvSOAR (pocket and void Surface of Amino Acid Residues), of comparing the protein surfaces of geometrically defined pockets and voids was developed. pvSOAR was able to detect previously unrecognized and novel functional relationships between surface features of proteins. In this study, pvSOAR is applied to several structural genomics proteins. We examined the surfaces of YecM, BioH, and RpiB from Escherichia coli as well as the CBS domains from inosine-5'-monosphate dehydrogenase from Streptococcus pyogenes, conserved hypothetical protein Ta549 from Thermoplasm acidophilum, and CBS domain protein mt1622 from Methanobacterium thermoautotrophicum with the goal to infer information about their biochemical function.     

Structural and enzymatic characterization of DR1281: A calcineurin-like phosphoesterase from Deinococcus radiodurans

We have determined the crystal structure of DR1281 from Deinococcus radiodurans. DR1281 is a protein of unknown function with over 170 homologs found in prokaryotes and eukaryotes. To elucidate the molecular function of DR1281, its crystal structure at 2.3 A resolution was determined and a series of biochemical screens for catalytic activity was performed. The crystal structure shows that DR1281 has two domains, a small alpha domain and a putative catalytic domain formed by a four-layered structure of two beta-sheets flanked by five alpha-helices on both sides. The small alpha domain interacts with other molecules in the asymmetric unit and contributes to the formation of oligomers. The structural comparison of the putative catalytic domain with known structures suggested its biochemical function to be a phosphatase, phosphodiesterase, nuclease, or nucleotidase. Structural analyses with its homologues also indicated that there is a dinuclear center at the interface of two domains formed by Asp8, Glu37, Asn38, Asn65, His148, His173, and His175. An absolute requirement of metal ions for activity has been proved by enzymatic assay with various divalent metal ions. A panel of general enzymatic assays of DR1281 revealed metal-dependent catalytic activity toward model substrates for phosphatases (p-nitrophenyl phosphate) and phosphodiesterases (bis-p-nitrophenyl phosphate). Subsequent secondary enzymatic screens with natural substrates demonstrated significant phosphatase activity toward phosphoenolpyruvate and phosphodiesterase activity toward 2',3'-cAMP. Thus, our structural and enzymatic studies have identified the biochemical function of DR1281 as a novel phosphatase/phosphodiesterase and disclosed key conserved residues involved in metal binding and catalytic activity.     

Structure and mechanism of ADP-ribose-1''-monophosphatase (Appr-1''-pase), a ubiquitous cellular processing enzyme

Appr-1'-pase, an important and ubiquitous cellular processing enzyme involved in the tRNA splicing pathway, catalyzes the conversion of ADP-ribose-1'monophosphate (Appr-1'-p) to ADP-ribose. The structures of the native enzyme from the yeast and its complex with ADP-ribose were determined to 1.9 A and 2.05 A, respectively. Analysis of the three-dimensional structure of this protein, selected as a target in a structural genomics project, reveals its putative function and provides clues to the catalytic mechanism. The structure of the 284-amino acid protein shows a two-domain architecture consisting of a three-layer alphabetaalpha sandwich N-terminal domain connected to a small C-terminal helical domain. The structure of Appr-1'-pase in complex with the product, ADP-ribose, reveals an active-site water molecule poised for nucleophilic attack on the terminal phosphate group. Loop-region residues Asn 80, Asp 90, and His 145 may form a catalytic triad.     

Structural analysis of the functional influence of the surface peptide Gtf-P1 on<Emphasis Type="Italic"> Streptococcus mutans</Emphasis> glucosyltransferase C activity

Glucosyltransferases (GtfB/C/D) in Streptococcus mutans are responsible for synthesizing water-insoluble and water-soluble glucans from sucrose and play very crucial roles in the formation of dental plaque. A monoclonal antibody against a 19-mer peptide fragment named Gtf-P1 was found in GtfC to reduce the enzyme activity to 50%. However, a similar experiment suggested almost unchanged activity in GtfD, despite of the very high sequence homology between the two enzymes. No further details are yet available to elucidate the biochemical mechanism responsible for such discrimination. For a better understanding of the catalytic behavior of these glucosyltransferases, structural and functional analyses were performed. First, the exact epitope was identified to specify the residue(s) required for monoclonal antibody recognition. The results suggest that the discrimination is determined solely by single residue substitution. Second, based on a combined sequence and secondary structure alignment against known crystal structure of segments from closely related proteins, a three-dimensional homology model for GtfC was built. Structural analysis for the region communicating between Gtf-P1 and the catalytic triad revealed the possibility for an "en bloc" movement of hydrophobic residues, which may transduce the functional influence on enzyme activity from the surface of molecule into the proximity of the active site. Figure Side chain interactions between Gtf-P1 and catalytic Asp-477 in GtfC. Calpha-tracing of GtfC with the two crucial peptides (Gtf-P1, orange; Gtf-P2, blue) and the catalytic triad residues ( red) highlighted to show their relative spatial organization. Side chains for the residues are also depicted according to their atom types. The structure is viewed with the barrel opening facing down     

Identify catalytic triads of serine hydrolases by support vector machines

The core of an enzyme molecule is its active site from the viewpoints of both academic research and industrial application. To reveal the structural and functional mechanism of an enzyme, one needs to know its active site; to conduct structure-based drug design by regulating the function of an enzyme, one needs to know the active site and its microenvironment as well. Given the atomic coordinates of an enzyme molecule, how can we predict its active site? To tackle such a problem, a distance group approach was proposed and the support vector machine algorithm applied to predict the catalytic triad of serine hydrolase family. The success rate by jackknife test for the 139 serine hydrolases was 85%, implying that the method is quite promising and may become a useful tool in structural bioinformatics.     

Crystal structure of yeast YHR049W/FSH1, a member of the serine hydrolase family

Yhr049w/FSH1 was recently identified in a combined computational and experimental proteomics analysis for the detection of active serine hydrolases in yeast. This analysis suggested that FSH1 might be a serine-type hydrolase belonging to the broad functional alphabeta-hydrolase superfamily. In order to get insight into the molecular function of this gene, it was targeted in our yeast structural genomics project. The crystal structure of the protein confirms that it contains a Ser/His/Asp catalytic triad that is part of a minimal alpha/beta-hydrolase fold. The architecture of the putative active site and analogies with other protein structures suggest that FSH1 may be an esterase. This finding was further strengthened by the unexpected presence of a compound covalently bound to the catalytic serine in the active site. Apparently, the enzyme was trapped with a reactive compound during the purification process.     

Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense

The crystal structure of a microbial transglutaminase from Streptoverticillium mobaraense has been determined at 2.4 A resolution. The protein folds into a plate-like shape, and has one deep cleft at the edge of the molecule. Its overall structure is completely different from that of the factor XIII-like transglutaminase, which possesses a cysteine protease-like catalytic triad. The catalytic residue, Cys(64), exists at the bottom of the cleft. Asp(255) resides at the position nearest to Cys(64) and is also adjacent to His(274). Interestingly, Cys(64), Asp(255), and His(274) superimpose well on the catalytic triad "Cys-His-Asp" of the factor XIII-like transglutaminase, in this order. The secondary structure frameworks around these residues are also similar to each other. These results imply that both transglutaminases are related by convergent evolution; however, the microbial transglutaminase has developed a novel catalytic mechanism specialized for the cross-linking reaction. The structure accounts well for the catalytic mechanism, in which Asp(255) is considered to be enzymatically essential, as well as for the causes of the higher reaction rate, the broader substrate specificity, and the lower deamidation activity of this enzyme.     

Structural and functional characterization of a novel phosphodiesterase from Methanococcus jannaschii

Methanococcus jannaschii MJ0936 is a hypothetical protein of unknown function with over 50 homologs found in many bacteria and Archaea. To help define the molecular (biochemical and biophysical) function of MJ0936, we determined its crystal structure at 2.4-A resolution and performed a series of biochemical screens for catalytic activity. The overall fold of this single domain protein consists of a four-layered structure formed by two beta-sheets flanked by alpha-helices on both sides. The crystal structure suggested its biochemical function to be a nuclease, phosphatase, or nucleotidase, with a requirement for some metal ions. Crystallization in the presence of Ni(2+) or Mn(2+) produced a protein containing a binuclear metal center in the putative active site formed by a cluster of conserved residues. Analysis of MJ0936 against a panel of general enzymatic assays revealed catalytic activity toward bis-p-nitrophenyl phosphate, an indicator substrate for phosphodiesterases and nucleases. Significant activity was also found with two other phosphodiesterase substrates, thymidine 5'-monophosphate p-nitrophenyl ester and p-nitrophenylphosphorylcholine, but no activity was found for cAMP or cGMP. Phosphodiesterase activity of MJ0936 had an absolute requirement for divalent metal ions with Ni(2+) and Mn(2+) being most effective. Thus, our structural and enzymatic studies have identified the biochemical function of MJ0936 as that of a novel phosphodiesterase.     

Catalytic promiscuity of Escherichia coli BioH esterase: Application in the synthesis of 3,4-dihydropyran derivatives

Enzymatic catalytic promiscuity has received increasing attention in the past decade. In this research, ten enzymes were investigated for the promiscuous activity in catalysis of the Michael addition-cyclization cascade reaction of p-nitrobenzalacetone with 1,3-cyclohexanedione to prepare 2-hydroxy-2-methyl-4-(4-nitrophenyl)-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one in anhydrous media, and control experiments were conducted to exclude false positive results. The highest yield (46.1%) was observed with Escherichia coli BioH esterase and the optimal reaction condition was: 1 mmol α,β-unsaturated ketone, 1 mmol 1,3-dicarbonyl compound, 20 mg E. coli BioH esterase, 20 ml N,N-dimethylformamide at 37 °C for 120 h. To preliminarily investigate the mechanism, site-directed mutagenesis was performed on the hydrolysis catalytic triad of BioH, and the results indicated “alternate-site enzyme promiscuity”. When a series of substituted benzalacetones and 1,3-cyclic diketones were used as the reactants, yields of up to 76.3% were achieved. These results imply the potential industrial application of E. coli BioH in the preparation of dihydropyran derivatives.     

The structure of arylamine N-acetyltransferase from Mycobacterium smegmatis--an enzyme which inactivates the anti-tubercular drug,isoniazid

Arylamine N-acetyltransferases which acetylate and inactivate isoniazid, an anti-tubercular drug, are found in mycobacteria including Mycobacterium smegmatis and Mycobacterium tuberculosis. We have solved the structure of arylamine N-acetyltransferase from M. smegmatis at a resolution of 1.7 A as a model for the highly homologous NAT from M. tuberculosis. The fold closely resembles that of NAT from Salmonella typhimurium, with a common catalytic triad and domain structure that is similar to certain cysteine proteases. The detailed geometry of the catalytic triad is typical of enzymes which use primary alcohols or thiols as activated nucleophiles. Thermal mobility and structural variations identify parts of NAT which might undergo conformational changes during catalysis. Sequence conservation among eubacterial NATs is restricted to structural residues of the protein core, as well as the active site and a hinge that connects the first two domains of the NAT structure. The structure of M. smegmatis NAT provides a template for modelling the structure of the M. tuberculosis enzyme and for structure-based ligand design as an approach to designing anti-TB drugs.     

A Francisella virulence factor catalyses an essential reaction of biotin synthesis

We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side‐chain. Expression of bioJ allows growth of an Escherichia coli bioH strain on biotin‐free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl‐ACP methyl ester carboxyl‐esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl‐ACP to pimeloyl‐ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel subclade of the α/β‐hydrolase family. Structure‐guided mapping combined with site‐directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted reaction in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence.     

Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome

A metagenomics approach is an efficient method of isolating novel and useful genes from uncultured microorganisms in diverse environments. In this research, a gene encoding a new esterase (EM2L8) was cloned and characterized from the metagenomic DNA library of a deep-sea sediment. The gene consisted of 804bp encoding a polypeptide of 267 amino acids with a molecular mass of 28,952. The deduced amino acid sequence showed similarities with the BioH of Kurthia, the 3-oxoadipate enol-lactonase of Haloarcula and the acyltransferase of Thermoanaerobacter, which feature identities of 38%, 32%, and 33%, respectively. Residues essential for esterase activity, such as pentapeptide (GXSXG) and catalytic triad sequences, were uncovered. While the protein was overproduced mainly as inclusion body at 37 degrees C, it was mainly produced as a soluble active enzyme at 18 degrees C. A zymogram analysis revealed that purified EM2L8 taken from the soluble fraction could hydrolyze tributyrin substrate. Furthermore, the protein from the inclusion body fraction also showed strong activity on gel, thus indicating that the protein was refolded during SDS-gel electrophoresis and the ensuing incubation period. When the inclusion body was mixed with some anionic detergent solutions and diluted with a non-detergent buffer, the insoluble EM2L8 refolded rapidly and recovered its full esterase activity. Although EM2L8 had an optimum temperature of 50-55 degrees C, its activation energy in the range of 10-40 degrees C was 8.34kcal/mol, indicating that it is a cold-adapted enzyme. Moreover, it was found to have an optimum pH of 10-11, thus revealing that it is an alkaline enzyme. In this paper, the new esterase EM2L8 buried in a deep-sea sediment became known on the surface and was characterized biochemically.