Catalytic Efficiency of Chitinase-D on Insoluble Chitinous Substrates Was Improved by Fusing Auxiliary Domains

Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and β-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP.     

Transglycosylation by Chitinase D from Serratia proteamaculans Improved through Altered Substrate Interactions

We describe the improvement of transglycosylation (TG) by chitinase D from Serratia proteamaculans (SpChiD). The SpChiD produced a smaller quantity of TG products for up to 90 min with 2 mm chitotetraose as the substrate and subsequently produced only hydrolytic products. Of the five residues targeted at the catalytic center, E159D resulted in substantial loss of both hydrolytic and TG activities. Y160A resulted in a product profile similar to SpChiD and a rapid turnover of substrate with slightly increased TG activity. The rest of the three mutants, M226A, Y228A, and R284A, displayed improved TG and decreased hydrolytic ability. Four of the five amino acid substitutions, F64W, F125A, G119S, and S116G, at the catalytic groove increased TG activity, whereas W120A completely lost the TG activity with a concomitant increase in hydrolysis. Mutation of Trp-247 at the solvent-accessible region significantly reduced the hydrolytic activity with increased TG activity. The mutants M226A, Y228A, F125A, S116G, F64W, G119S, R284A, and W247A accumulated approximately double the concentration of TG products like chitopentaose and chitohexaose, compared with SpChiD. The double mutant E159D/F64W regained the activity with accumulation of 6.0% chitopentaose at 6 h, similar to SpChiD at 30 min. Loss of chitobiase activity was unique to Y228A. Substitution of amino acids at the catalytic center and/or groove substantially improved the TG activity of SpChiD, both in terms of the quantity of TG products produced and the extended duration of TG activity.     

Molecular modeling and docking characterization of CzR1, a CC-NBS-LRR R-gene from Curcuma zedoaria Loeb. that confers resistance to Pythium aphanidermatum

Plant NBS-LRR R-genes recognizes several pathogen associated molecular patterns (PAMPs) and limit pathogen infection through a multifaceted defense response. CzR1, a coiled-coil-nucleotide-binding-site-leucine-rich repeat R-gene isolated from Curcuma zedoaria L exhibit constitutive resistance to different strains of P. aphanidermatum. Majority of the necrotrophic oomycetes are characterized by the presence of carbohydrate PAMPs β-glucans in their cell walls which intercat with R-genes. In the present study, we predicted the 3D (three dimensional) structure of CzR1 based on homology modeling using the homology module of Prime through the Maestro interface of Schrodinger package ver 2.5. The docking investigation of CzR1 with β-glucan using the Glide software suggests that six amino acid residues, Ser186, Glu187, Ser263, Asp264, Asp355 and Tyr425 act as catalytic residues and are involved in hydrogen bonding with ligand β-(1,3)-D-Glucan. The calculated distance between the carboxylic oxygen atoms of Glu187–Asp355 pair is well within the distance of 5Å suggesting a positive glucanase activity of CzR1. Elucidation of these molecular characteristics will help in in silico screening and understanding the structural basis of ligand binding to CzR1 protein and pave new ways towards a broad spectrum rhizome rot resistance development in the cultivated turmeric.     

Reversing Evolution: Re-establishing Obligate Metal Ion Dependence in a Metal-independent KDO8P Synthase

Two distinct groups of 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS), a key enzyme of cell-wall biosynthesis, differ by their requirement for a divalent metal ion for enzymatic activity. The unique difference between these groups is the replacement of the metal-binding Cys by Asn. Substitution of just this Asn for a Cys in metal-independent KDO8PS does not create the obligate metal-ion dependency of natural metal-dependent enzymes. We describe how three or four mutations of the metal-independent KDO8PS from Neisseria meningitidis produce a fully functional, obligately metal-dependent KDO8PS. For the substitutions Asn23Cys, Asp247Glu (this Asp binds to the metal ion in all metal-dependent KDO8PS) and Pro249Ala, and for double and triple combinations, mutant enzymes that contained Cys in place of Asn showed an increase in activity in the presence of divalent metal ions. However, combining these mutations with substitution by Ser of the Cys residue in the conserved ²⁴⁶CysAspGlyPro²⁴⁹ motif of metal-independent KDO8PS created enzymes with obligate metal dependency. The quadruple mutant (Asn23Cys/Cys246Ser/Asp247Glu/Pro249Ala) showed comparable activity to wild-type enzymes only in the presence of metal ions, with maximum activity with Cd²⁺, the metal ion that is strongly inhibitory at micromolar concentrations for the wild-type enzyme. In the absence of metal ions, activity was barely detectable for this quadruple mutant or for triple mutants bearing both Cys246Ser and Asn23Cys mutations. The structures of NmeKDO8PS and its Asn23Cys/Asp247Glu/Pro249Ala and quadruple mutants at pH 4.6 were characterized at resolutions better than 1.85 Å. Aged crystals of the Asn23Cys/Asp247Glu/Pro249Ala mutant featured a Cys23-Cys246 disulfide linkage, explaining the spectral bleaching observed when this mutant was incubated with Cu²⁺. Such bleaching was not observed for the quadruple mutant. Reverse evolution to a fully functional obligately metal-dependent KDO8PS has been achieved with just three directed mutations for enzymes that have, at best, 47% identity between metal-dependent and metal-independent pairs.     

Distinct conformation-mediated functions of an active site loop in the catalytic reactions of NAD-dependent D-lactate dehydrogenase and formate dehydrogenase

The three-dimensional structures of NAD-dependent D-lactate dehydrogenase (D-LDH) and formate dehydrogenase (FDH), which resemble each other, imply that the two enzymes commonly employ certain main chain atoms, which are located on corresponding loop structures in the active sites of the two enzymes, for their respective catalytic functions. These active site loops adopt different conformations in the two enzymes, a difference likely attributable to hydrogen bonds with Asn97 and Glu141, which are also located at equivalent positions in D-LDH and FDH, respectively. X-ray crystallography at 2.4-A resolution revealed that replacement of Asn97 with Asp did not markedly change the overall protein structure but markedly perturbed the conformation of the active site loop in Lactobacillus pentosus D-LDH. The Asn97-->Asp mutant D-LDH exhibited virtually the same k(cat), but about 70-fold higher K(M) value for pyruvate than the wild-type enzyme. For Paracoccus sp. 12-A FDH, in contrast, replacement of Glu141 with Gln and Asn induced only 5.5- and 4.3-fold increases in the K(M) value, but 110 and 590-fold decreases in the k(cat) values for formate, respectively. Furthermore, these mutant FDHs, particularly the Glu141-->Asn enzyme, exhibited markedly enhanced catalytic activity for glyoxylate reduction, indicating that FDH is converted to a 2-hydroxy-acid dehydrogenase on the replacement of Glu141. These results indicate that the active site loops play different roles in the catalytic reactions of D-LDH and FDH, stabilization of substrate binding and promotion of hydrogen transfer, respectively, and that Asn97 and Glu141, which stabilize suitable loop conformations, are essential elements for proper loop functioning.     

Molecular modeling and analysis of human and plant endo-β-N-acetyl- glucosaminidases for mutations effects on function

Endo- β-N-acetylgucosaminidases (ENGases) are the enzymes that catalyze both hydrolysis and transglycosylation reactions. It is of interest to study ENGases because of their ability to synthesize glycopeptides. Homology models of Human, Arabidopsis thaliana and Sorghum ENGases were developed and their active sites marked based on information available from Arthrobacter protophormiae (PDB ID: 3FHQ) ENGase. Further, these models were docked with the natural substrate GlcNAc-Asn and the inhibitor Man₃GlcNAc-thiazoline. The catalytic triad of Asn, Glu and Tyr (N171, E173 and Y205 of bacteria) were found to be conserved across the phyla. The crucial Y299F mutation showing 3 times higher transglycosylation activity than in wild type Endo-A is known. The hydrolytic activity remained unchanged in bacteria, while the transglycosylation activity increased. This Y to F change is found to be naturally evolved and should be attributing higher transglycosylation rates in human and Arabidopsis thaliana ENGases. Ligand interactions Ligplots revealed the interaction of amino acids with hydrophobic side chains and polar uncharged side chain amino acids. Thus, structure based molecular model-ligand interactions provide insights into the catalytic mechanism of ENGases and assist in the rational engineering of ENGases.     

Implication from the predicted docked interaction of sigma H and exploration of its interaction with RNA polymerase in Mycobacterium tuberculosis

M. tuberculosis is adapted to remain active in the extreme environmental condition due to the presence of atypical sigma factors commonly called extra cytoplasmic function (ECF) sigma factors. Among the 13 sigma factors of M. tuberculosis, 10 are regarded as the ECF sigma factor that exerts their attributes in various stress response. Therefore it is of interest to describe the structural prediction of one of the ECF sigma factors, sigma H (SigH), involved in oxidative and heat stress having interaction with the β׳ subunit of M. tuberculosis. RNA polymerase (Mtb-RNAP). The model of Mtb-SigH was build using the commercial package of Discovery Studio version 2.5 from Accelerys (San Diego, CA, USA) containing the inbuilt MODELER module and that of β׳ subunit of Mtb-RNAP using Phyre Server. Further, the protein models were docked using the fully automated web tool ClusPro (cluspro.bu.edu/login.php). Mtb-SigH is a triple helical structure having a putative DNA-binding site and the β׳ subunit of MtbRNAP consists of 18-beta sheets and 22 helices. The SigH-Mtb-RNAP β׳ interaction studies showed that Arg26, Gln19 andAsp18, residues of SigH protein are involved in binding with Arg137, Gln140, Arg152, Asn133 and Asp144 of β׳ subunit of Mtb-RNAP. The predicted model helps to explore the molecular mechanism in the control of gene regulation with a novel unique target for potential new generation inhibitor.     

Amino acid substitution at the substrate-binding subsite alters the specificity of the Phanerochaete chrysosporium cellobiose dehydrogenase

The active site of cellobiose dehydrogenase from Phanerochaete chrysosporium is composed of two subsites, a catalytic C subsite and a substrate-binding B subsite. Based on the crystal structure of the enzyme with a cellobiose analogue, residue Glu279 was selected for site-directed mutagenesis studies. Substitution of Glu279 to Ala, Asn, and Asp had no effect on the expression of the protein in Pichia pastoris but completely abolished its enzymatic activity. Substitution of Glu279 to Gln drastically altered the enzyme’s substrate specificity. While the wild-type cellobiose dehydrogenase efficiently oxidizes cellobiose and lactose, the Glu279Gln mutant retained most of its activity with cellobiose but was completely inactive with lactose. We generated structural models of the active site interacting with cellobiose and lactose to provide an interpretation of these results.     

Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate

Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast—Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PP_i. Crystal structures of metal-free Duf89 and Co²⁺-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl₂ and NaF in the presence of divalent cations Co²⁺, Ni²⁺, or Zn²⁺ generated covalent Duf8901-(Asp248)–beryllium trifluoride (BeF₃)•Co²⁺, Duf8901-(Asp248)–BeF₃•Ni²⁺, or Duf8901-(Asp248)–BeF₃•Zn²⁺ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)–phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF₃•Co²⁺ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)–AlF₄–OH₂•Co²⁺ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.     

Aldehyde dehydrogenase enzyme ALDH3H1 from Arabidopsis thaliana: Identification of amino acid residues critical for cofactor specificity

The cofactor-binding site of the NAD⁺-dependent Arabidopsis thaliana aldehyde dehydrogenase ALDH3H1 was analyzed to understand structural features determining cofactor-specificity. Homology modeling and mutant analysis elucidated important amino acid residues. Glu149 occupies a central position in the cofactor-binding cleft, and its carboxylate group coordinates the 2′- and 3′-hydroxyl groups of the adenosyl ribose ring of NAD⁺ and repels the 2′-phosphate moiety of NADP⁺. If Glu149 is mutated to Gln, Asp, Asn or Thr the binding of NAD⁺ is altered and rendered the enzyme capable of using NADP⁺. This change is attributed to a weaker steric hindrance and elimination of the electrostatic repulsion force of the 2′-phosphate of NADP⁺. Simultaneous mutations of Glu149 and Ile200, which is situated opposite of the cofactor binding cleft, improved the enzyme efficiency with NADP⁺. The double mutant ALDH3H1_{Glu149Thr/Ile200Val} showed a good catalysis with NADP⁺. Subsequently a triple mutation was generated by replacing Val178 by Arg in order to create a “closed” cofactor binding site. The cofactor specificity was shifted even further in favor of NADP⁺, as the mutant ALDH3H1_{E149T/V178R/I200V} uses NADP⁺ with almost 7-fold higher catalytic efficiency compared to NAD⁺. Our experiments suggest that residues occupying positions equivalent to 149, 178 and 200 constitute a group of amino acids in the ALDH3H1 protein determining cofactor affinity.     

Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga

Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light-dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 Å resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the “late proton release”.     

Exome Sequencing Identifies GNB4 Mutations as a Cause of Dominant Intermediate Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of inherited neuropathies. Mutations in approximately 45 genes have been identified as being associated with CMT. Nevertheless, the genetic etiologies of at least 30% of CMTs have yet to be elucidated. Using a genome-wide linkage study, we previously mapped a dominant intermediate CMT to chromosomal region 3q28–q29. Subsequent exome sequencing of two affected first cousins revealed heterozygous mutation c.158G>A (p.Gly53Asp) in GNB4, encoding guanine-nucleotide-binding protein subunit beta-4 (Gβ₄), to cosegregate with the CMT phenotype in the family. Further analysis of GNB4 in an additional 88 unrelated CMT individuals uncovered another de novo mutation, c.265A>G (p.Lys89Glu), in this gene in one individual. Immunohistochemistry studies revealed that Gβ₄ was abundant in the axons and Schwann cells of peripheral nerves and that expression of Gβ₄ was significantly reduced in the sural nerve of the two individuals carrying the c.158G>A (p.Gly53Asp) mutation. In vitro studies demonstrated that both the p.Gly53Asp and p.Lys89Glu altered proteins impaired bradykinin-induced G-protein-coupled-receptor (GPCR) signaling, which was facilitated by the wild-type Gβ₄. This study identifies GNB4 mutations as a cause of CMT and highlights the importance of Gβ₄-related GPCR signaling in peripheral-nerve function in humans.     

A Pathogenesis Related Protein,VpPR-10.1, from Vitis pseudoreticulata: An Insight of Its Mode of Antifungal Activity

Previously, VpPR-10.1 was isolated and characterized from a cDNA library of a fungus-resistant accession of Chinese wild grape (Vitis pseudoreticulata). We found that expression of VpPR-10.1 is affected by the fungal pathogen Erysiphe necator. To investigate the biochemical basis of the nuclease activity of VpPR-10.1 and its role in antifungal resistance, we generated recombinant VpPR-10.1 as well as site-directed mutations targeting three conserved amino acid residues among plant PR-10 s: Lys55, Glu149, and Tyr151. We showed that wild-type recombinant VpPR-10.1 exhibits both RNase and DNase activities. Mutant VpPR10.1-Y151H essentially retained all these activities. In contrast, VpPR10.1-K55N, where Lys55 in the P-loop region is mutated to Asn, and VpPR10.1-E149G, where Glu149 is mutated to Gly, lost their nuclease activity, indicating that both residues play a critical role in catalyzing RNA and DNA degradation. Furthermore, VpPR10.1 and VpPR10.1-Y151H inhibited the growth of the cultured fungal pathogen Alternaria alternate. Through transient expression in grapevine, we also demonstrated that VpPR10.1-K55N and VpPR10.1-E149G compromised resistance to E. necator. Finally, we further found that VpPR-10.1 can lead to programmed cell death and DNA degradation when incubated with tobacco BY-2 suspension cells. We show here that Lys55 and Glu149, but not Tyr151, are required for the RNase, DNase and antifungal activities of VpPR-10.1. The strong correlation between the level of VpPR-10.1 nuclease activity and its antifungal property indicates that the former is the biochemical basis for the latter. Taken together, our experiments revealed that VpPR-10.1 is critical in mediating fungal resistance in grape, potentially playing a dual role by degrading pathogen RNA and inducing programmed death of host cells.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Reaction mechanism of tetrathionate hydrolysis based on the crystal structure of tetrathionate hydrolase from Acidithiobacillus ferrooxidans

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron‐ and sulfur‐oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af‐Tth) was determined by X‐ray crystallography to a resolution of 1.95 Å. Af‐Tth is a homodimer, and its monomer structure exhibits an eight‐bladed β‐propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the β‐propeller region. We observed unexplained electron densities in this cavity of the substrate‐soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site‐specific Af‐Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af‐Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step.     

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ¹-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na⁺-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means.     

Specificity and mutational analysis of the metal-dependent 3-deoxy-d-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans

3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenol pyruvate and d-arabinose 5-phosphate to generate KDO8P. This reaction is part of the biosynthetic pathway to 3-deoxy-d-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Two distinct groups of KDO8PSs exist, differing by the absolute requirement of a divalent metal ion. In this study Acidithiobacillus ferrooxidans KDO8PS has been expressed and purified and shown to require a divalent metal ion, with Mn²⁺, Co²⁺ and Cd²⁺ (in decreasing order) being able to restore activity to metal-free enzyme. Cd²⁺ significantly enhanced the stability of the enzyme, raising the T_m by 14 °C. d-Glucose 6-phosphate and d-erythrose 4-phosphate were not substrates for A. ferrooxidans KDO8PS, whereas 2-deoxy-d-ribose 5-phosphate was a poor substrate and there was negligible activity with d-ribose 5-phosphate. The ²⁴³AspGlyPro²⁴⁵ motif is absolutely conserved in the metal-independent group of synthases, but the Gly and Pro sites are variable in the metal-dependent enzymes. Substitution of the putative metal-binding Asp243 to Ala in A. ferrooxidans KDO8PS gave inactive enzyme, whereas substitutions Asp243Glu or Pro245Ala produced active enzymes with altered metal-dependency profiles. Prior studies indicated that exchange of a metal-binding Cys for Asn converts metal-dependent KDO8P synthase into a metal-independent form. Unexpectedly, this mutation in A. ferrooxidans KDO8P synthase (Cys21Asn) gave inactive enzyme. This finding, together with modest activity towards 2-deoxy-d-ribose 5-phosphate suggests similarities between the A. ferrooxidans KDO8PS and the related metal-dependent 3-deoxy-d-arabino-heptulosonate phosphate synthase, and highlights the importance of the AspGlyPro loop in positioning the substrate for effective catalysis in all KDO8P synthases.     

Crystal structure of peptidyl-tRNA hydrolase from mycobacterium smegmatis reveals novel features related to enzyme dynamics

Peptidyl-tRNA hydrolase from Mycobacterium smegmatis is a single domain 21 kDa protein involved in the hydrolysis of prematurely produced peptidyl-tRNAs to ensure the viability of cells in bacteria, thus making it a potentially important drug target. In order to aid the development of potent drugs for controlling bacterial infections, the three-dimensional structure of peptidyl-tRNA hydrolase from Mycobacterium smegmatis has been determined. The protein adopts a compact α/β globular fold with a twisted β-sheet surrounded by α-helices. The functionally important C-terminal stretch has been unambiguously modeled for the first time in the unliganded structure of peptidyl-tRNA hydrolase. The segment, Gly138 - Val150 is mobile because it lacks significant interactions with the rest of the protein molecule. This conformational flexibility is reflected through different values of distances between a reference atom Ala147 C^α of the segment Gly138 - Val150 to Gly114 C^α from another segment from opposite side of the substrate binding channel in Mycobacterium smegmatis (7.8 Ǻ), Mycobacterium tuberculosis (9.5 Ǻ) and Escherichia coli (11.8 Ǻ). Similarly, the conformation of loop Gly109 - Gly117 with respect to another loop Asp95 - Asp100 also shows variability of the substrate binding cleft as the distance between Asp98 O^δ2 to Gly113 C^α in Mycobacterium smegmatis is 4.5 Ǻ while the corresponding distances in Mycobacterium tuberculosis and Escherichia coli are 3.1 Ǻ and 6.7 Ǻ respectively. The hydrogen bonded interactions between Asn116, His22 and Asp95 indicate a stereochemically favorable arrangement of these residues for catalytic action.