cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

Effect of N-Guanyl Modifications on Early Steps in Catalysis of Polymerization by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W

Translesion DNA polymerases are more efficient at bypass of many DNA adducts than replicative polymerases. Previous work with the translesion polymerase Sulfolobus solfataricus Dpo4 showed a decrease in catalytic efficiency during bypass of bulky N²-alkyl guanine (G) adducts with N²-isobutylG showing the largest effect, decreasing ∼ 120-fold relative to unmodified deoxyguanosine (Zhang, H., Eoff, R. L., Egli, M., Guengerich, F. P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translation synthesis past bulky N²-alkylguanine adducts. J. Biol. Chem. 2009; 284: 3563-3576). The effect of adduct size on individual catalytic steps has not been easy to decipher because of the difficulty of distinguishing early noncovalent steps from phosphodiester bond formation. We developed a mutant with a single Trp (T239W) to monitor fluorescence changes associated with a conformational change that occurs after binding a correct 2′-deoxyribonucleoside triphosphate (Beckman, J. W., Wang, Q., Guengerich, F. P. Kinetic analysis of nucleotide insertion by a Y-family DNA polymerase reveals conformational change both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 2008; 283: 36711-36723) and, in the present work, utilized this approach to monitor insertion opposite N²-alkylG-modified oligonucleotides. We estimated maximal rates for the forward conformational step, which coupled with measured rates of product formation yielded rate constants for the conformational step (both directions) during insertion opposite several N²-alkylG adducts. With the smaller N²-alkylG adducts, the conformational rate constants were not changed dramatically (<  3-fold), indicating that the more sensitive steps are phosphodiester bond formation and partitioning into inactive complexes. With the larger adducts (≥  (2-naphthyl)methyl), the absence of fluorescence changes suggests impaired ability to undergo an appropriate conformational change, consistent with previous structural work.     

Structural and biochemical characterization of the broad substrate specificity of Bacteroides thetaiotaomicron commensal sialidase

Sialidases release the terminal sialic acid residue from a wide range of sialic acid-containing polysaccharides. Bacteroides thetaiotaomicron, a symbiotic commensal microbe, resides in and dominates the human intestinal tract. We characterized the recombinant sialidase from B. thetaiotaomicron (BTSA) and demonstrated that it has broad substrate specificity with a relative activity of 97, 100 and 64 for 2,3-, 2,6- and 2,8-linked sialic substrates, respectively. The hydrolysis activity of BTSA was inhibited by a transition state analogue, 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid, by competitive inhibition with a K_i value of 35 μM. The structure of BSTA was determined at a resolution of 2.3 Å. This structure exhibited a unique carbohydrate-binding domain (CBM) at its N-terminus (a.a. 23–190) that is adjacent to the catalytic domain (a.a. 191–535). The catalytic domain has a conserved arginine triad with a wide-open entrance for the substrate that exposes the catalytic residue to the surface. Unlike other pathogenic sialidases, the polysaccharide-binding site in the CBM is near the active site and possibly holds and positions the polysaccharide substrate directly at the active site. The structural feature of a wide substrate-binding groove and closer proximity of the polysaccharide-binding site to the active site could be a unique signature of the commensal sialidase BTSA and provide a molecular basis for its pharmaceutical application.     

Crystal structures of an O-like blue form and an anion-free yellow form of pharaonis halorhodopsin

Halorhodopsin from Natronomonas pharaonis (pHR) was previously crystallized into a monoclinic space group C2, and the structure of the chloride-bound purple form was determined. Here, we report the crystal structures of two chloride-free forms of pHR, that is, an O-like blue form and an M-like yellow form. When the C2 crystal was soaked in a chloride-free alkaline solution, the protein packing was largely altered and the yellow form containing all-trans retinal was generated. Upon neutralization, this yellow form was converted into the blue form. From structural comparison of the different forms of pHR, it was shown that the removal of a chloride ion from the primary binding site (site I), which is located between the retinal Schiff base and Thr126, is accompanied by such a deformation of helix C that the side chain of Thr126 moves toward helix G, leading to a significant shrinkage of site I. A large structural change is also induced in the chloride uptake pathway, where a flip motion of the side chain of Glu234 is accompanied by large movements of the surrounding aromatic residues. Irrespective of different charge distributions at the active site, there was no large difference in the structures of the yellow form and the blue form. It is shown that the yellow-to-purple transition is initiated by the entrance of one water and one HCl to the active site, where the proton and the chloride ion in HCl are transferred to the Schiff base and site I, respectively.     

X-ray structures of Bacillus pallidusd-arabinose isomerase and its complex with l-fucitol

d-Arabinose isomerase (d-AI), also known as l-fucose isomerase (l-FI), catalyzes the aldose–ketose isomerization of d-arabinose to d-ribulose, and l-fucose to l-fuculose. Bacillus pallidus (B. pallidus) d-AI can catalyze isomerization of d-altrose to d-psicose, as well as d-arabinose and l-fucose. Three X-ray structures of B. pallidusd-AI in complexes with 2-methyl-2,4-pentadiol, glycerol and an inhibitor, l-fucitol, were determined at resolutions of 1.77, 1.60 and 2.60 Å, respectively. B. pallidusd-AI forms a homo-hexamer, and one subunit has three domains of almost equal size; two Rossmann fold domains and a mimic of the (β/α) barrel fold domain. A catalytic metal ion (Mn²⁺) was found in the active site coordinated by Glu342, Asp366 and His532, and an additional metal ion was found at the channel for the passage of a substrate coordinated by Asp453. The X-ray structures basically supported the ene-diol mechanism for the aldose–ketose isomerization by B. pallidusd-AI, as well as Escherichia coli (E. coli) l-FI, in which Glu342 and Asp366 facing each other at the catalytic metal ion transfer a proton from C2 to C1 and O1 to O2, acting as acid/base catalysts, respectively. However, considering the ionized state of Asp366, the catalytic reaction also possibly occurs through the negatively charged ene-diolate intermediate stabilized by the catalytic metal ion. A structural comparison with E. colil-FI showed that B. pallidusd-AI possibly interconverts between “open” and “closed” forms, and that the additional metal ion found in B. pallidusd-AI may help to stabilize the channel region.     

Non-lipolytic and lipolytic sequence-related carboxylesterases: A comparative study of the structure–function relationships of rabbit liver esterase 1 and bovine pancreatic bile-salt-activated lipase

To differentiate esterases from lipases at the structure–function level, we have compared the kinetic properties and structural features of sequence-related esterase 1 from rabbit liver (rLE) and bile-salt-activated lipase from bovine pancreas (bBAL). In contrast to rLE, bBAL hydrolyses water-insoluble medium and long chain esters as vinyl laurate, trioctanoin and olive oil. Conversely, rLE and bBAL are both active on water-soluble short chain esters as vinyl acetate, vinyl propionate, vinyl butyrate, tripropionin, tributyrin and p-nitrophenyl butyrate. However, the enzymes show distinctive kinetic behaviours. rLE displays maximal activity at low substrate concentration, below the critical micelle concentration, whereas bBAL acts preferencially on emulsified esters, at concentration exceeding the solubility limit. Comparison of the 3D structures of rLE and bBAL shows, in particular, that the peptide loop at positions 116–123 in bBAL is deleted in rLE. This peptide segment interacts with a bile salt molecule thus inducing a conformational transition which gives access to the active site. Inhibition studies and manual docking of a bulky ester molecule as vinyl laurate in the catalytic pocket of rLE and bBAL show that the inability of the esterase to hydrolyse large water-insoluble esters is not due to steric hindrance. It is hypothesized that esterases lack specific hydrophobic structures involved both in the stabilization of the lipase–lipid adsorption complex at interfaces and in the spontaneous transfer of a single substrate molecule from interface to the catalytic site.     

Fine tuning of the active site modulates specificity in the interaction of O-acetylserine sulfhydrylase isozymes with serine acetyltransferase

O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of l-cysteine in the last step of the reductive sulfate assimilation pathway in microorganisms. Its activity is inhibited by the interaction with serine acetyltransferase (SAT), the preceding enzyme in the metabolic pathway. Inhibition is exerted by the insertion of SAT C-terminal peptide into the OASS active site. This action is effective only on the A isozyme, the prevalent form in enteric bacteria under aerobic conditions, but not on the B-isozyme, the form expressed under anaerobic conditions. We have investigated the active site determinants that modulate the interaction specificity by comparing the binding affinity of thirteen pentapeptides, derived from the C-terminal sequences of SAT of the closely related species Haemophilus influenzae and Salmonella typhimurium, towards the corresponding OASS-A, and towards S. typhimurium OASS-B. We have found that subtle changes in protein active sites have profound effects on protein–peptide recognition. Furthermore, affinity is strongly dependent on the pentapeptide sequence, signaling the relevance of P3–P4–P5 for the strength of binding, and P1–P2 mainly for specificity. The presence of an aromatic residue at P3 results in high affinity peptides with K_diss in the micromolar and submicromolar range, regardless of the species. An acidic residue, like aspartate at P4, further strengthens the interaction and results in the higher affinity ligand of S. typhimurium OASS-A described to date. Since OASS knocked-out bacteria exhibit a significantly decreased fitness, this investigation provides key information for the development of selective OASS inhibitors, potentially useful as novel antibiotic agents.     

High tolerance to mutations in a Chlamydia trachomatis peptide deformylase loop

AIM: To determine if and how a loop region in the peptide deformylase (PDF) of Chlamydia trachomatis regulates enzyme function.METHODS: Molecular dynamics simulation was used to study a structural model of the chlamydial PDF (cPDF) and predict the temperature factor per residue for the protein backbone atoms. Site-directed mutagenesis was performed to construct cPDF variants. Catalytic properties of the resulting variants were determined by an enzyme assay using formyl-Met-Ala-Ser as a substrate.RESULTS: In silico analysis predicted a significant increase in atomic motion in the DGELV sequence (residues 68-72) of a loop region in a cPDF mutant, which is resistant to PDF inhibitors due to two amino acid substitutions near the active site, as compared to wild-type cPDF. The D68R and D68R/E70R cPDF variants demonstrated significantly increased catalytic efficiency. The E70R mutant showed only slightly decreased efficiency. Although deletion of residues 68-72 resulted in a nearly threefold loss in substrate binding, this deficiency was compensated for by increased catalytic efficiency.CONCLUSION: Movement of the DGELV loop region is involved in a rate-limiting conformational change of the enzyme during catalysis. However, there is no stringent sequence requirement for this region for cPDF enzyme activity.     

The role of short-range Cys171-Cys178 disulfide bond in maintaining cutinase active site integrity: A molecular dynamics simulation

Understanding structural determinants in enzyme active site integrity can provide a good knowledge to design efficient novel catalytic machineries. Fusarium solani pisi cutinase with classic triad Ser-His-Asp is a promising enzyme to scrutinize these structural determinants. We performed two MD simulations: one, with the native structure, and the other with the broken Cys171-Cys178 disulfide bond. This disulfide bond stabilizes a turn in active site on which catalytic Asp175 is located. Functionally important H-bonds and atomic fluctuations in catalytic pocket have been changed. We proposed that this disulfide bond within active site can be considered as an important determinant of cutinase active site structural integrity.     

Oligomeric State in the Crystal Structure of Modular FAD Synthetase Provides Insights into Its Sequential Catalysis in Prokaryotes

The crystal structure of the modular flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes has been solved at 1.95 Å resolution. The structure of C. ammoniagenes FAD synthetase presents two catalytic modules—a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide (FMN) adenylyltransferase activity—that are responsible for the synthesis of FAD from riboflavin in two sequential steps. In the monomeric structure, the active sites from both modules are placed 40 Å away, preventing the direct transfer of the product from the first reaction (FMN) to the second catalytic site, where it acts as substrate. Crystallographic and biophysical studies revealed a hexameric assembly formed by the interaction of two trimers. Each trimer presents a head-tail configuration, with FMN adenylyltransferase and riboflavin kinase modules from different protomers approaching the active sites and allowing the direct transfer of FMN. Experimental results provide molecular-level evidences of the mechanism of the synthesis of FMN and FAD in prokaryotes in which the oligomeric state could be involved in the regulation of the catalytic efficiency of the modular enzyme.     

The molecular structure of epoxide hydrolase B from Mycobacterium tuberculosis and its complex with a urea-based inhibitor

Mycobacterium tuberculosis (Mtb), the intracellular pathogen that infects macrophages primarily, is the causative agent of the infectious disease tuberculosis in humans. The Mtb genome encodes at least six epoxide hydrolases (EHs A to F). EHs convert epoxides to trans-dihydrodiols and have roles in drug metabolism as well as in the processing of signaling molecules. Herein, we report the crystal structures of unbound Mtb EHB and Mtb EHB bound to a potent, low-nanomolar (IC₅₀ ≈ 19 nM) urea-based inhibitor at 2.1 and 2.4 Å resolution, respectively. The enzyme is a homodimer; each monomer adopts the classical α/β hydrolase fold that composes the catalytic domain; there is a cap domain that regulates access to the active site. The catalytic triad, comprising Asp104, His333 and Asp302, protrudes from the catalytic domain into the substrate binding cavity between the two domains. The urea portion of the inhibitor is bound in the catalytic cavity, mimicking, in part, the substrate binding; the two urea nitrogen atoms donate hydrogen bonds to the nucleophilic carboxylate of Asp104, and the carbonyl oxygen of the urea moiety receives hydrogen bonds from the phenolic oxygen atoms of Tyr164 and Tyr272. The phenolic oxygen groups of these two residues provide electrophilic assistance during the epoxide hydrolytic cleavage. Upon inhibitor binding, the binding-site residues undergo subtle structural rearrangement. In particular, the side chain of Ile137 exhibits a rotation of around 120° about its C^α-C^β bond in order to accommodate the inhibitor. These findings have not only shed light on the enzyme mechanism but also have opened a path for the development of potent inhibitors with good pharmacokinetic profiles against all Mtb EHs of the α/β type.     

Crystal structures and mutagenesis of sucrose hydrolase from Xanthomonas axonopodis pv. glycines: insight into the exclusively hydrolytic amylosucrase fold

Neisseria polysaccharea amylosucrase (NpAS), a transglucosidase of glycoside hydrolase family 13, is a hydrolase and glucosyltransferase that catalyzes the synthesis of amylose-like polymer from a sucrose substrate. Recently, an NpAS homolog from Xanthomonas axonopodis pv. glycines was identified as a member of the newly defined carbohydrate utilization locus that regulates the utilization of plant sucrose in phytopathogenic bacteria. Interestingly, this enzyme is exclusively a hydrolase and not a glucosyltransferase; it is thus known as sucrose hydrolase (SUH). Here, we elucidated the novel functional features of SUH using X-ray crystallography and site-directed mutagenesis. Four different crystal structures of SUH, including the SUH-Tris and the SUH-sucrose and SUH-glucose complexes, represent structural snapshots along the catalytic reaction coordinate. These structures show that SUH is distinctly different from NpAS in that ligand-induced conformational changes in SUH cause the formation of a pocket-shaped active site and in that SUH lacks the three arginine residues found in the NpAS active site that appear to be crucial for NpAS glucosyltransferase activity. Mutation of SUH to insert these arginines failed to confer glucosyltransferase activity, providing evidence that its enzymatic activity is limited to sucrose hydrolysis by its pocket-shaped active site and the identity of residues in the vicinity of the active site.