Putative Catalytic Site Research Articles

Structural Characterization of a Lectin from the Mushroom Marasmius oreades in Complex with the Blood Group B Trisaccharide and Calcium

MOA (Marasmius oreades agglutinin), a lectin isolated from fruiting bodies of the mushroom M. oreades, specifically binds nonreducing terminal Galalpha(1,3)Gal carbohydrates, such as that which occurs in the xenotransplantation epitope Galalpha(1,3)Galbeta(1,4)GlcNAc and the branched blood group B determinant Galalpha(1,3)[Fucalpha(1,2)]Gal. Here, we present the crystal structure of MOA in complex with the blood group B trisaccharide solved at 1.8 A resolution. To our knowledge, this is the first blood-group-B-specific structure reported in complex with a blood group B determinant. The carbohydrate ligand binds to all three binding sites of the N-terminal beta-trefoil domain. Also, in this work, Ca(2+) was included in the crystals, and binding of Ca(2+) to the MOA homodimer altered the conformation of the C-terminal domain by opening up the cleft containing a putative catalytic site.

Biochemical Analysis of a β- d -Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

Prevotella ruminicola 23 is an obligate anaerobic bacterium in the phylum Bacteroidetes that contributes to hemicellulose utilization within the bovine rumen. To gain insight into the cellular machinery that this organism elaborates to degrade the hemicellulosic polymer xylan, we identified and cloned a gene predicted to encode a bifunctional xylanase-ferulic acid esterase (xyn10D-fae1A) and expressed the recombinant protein in Escherichia coli. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-beta-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 beta-D-glucosidase gene, with a novel PA14-like insertion sequence, was identified two genes downstream of xyn10D-fae1A. Biochemical analyses of the purified recombinant protein revealed that the putative beta-D-glucosidase has activity for pNP-beta-D-xylopyranoside, pNP-alpha-L-arabinofuranoside, and xylo-oligosaccharides; thus, the gene was designated xyl3A. When incubated in combination with Xyn10D-Fae1A, Xyl3A improved the release of xylose monomers from a hemicellulosic xylan substrate, suggesting that these two enzymes function synergistically to depolymerize xylan. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. When a mutation was introduced into the putative catalytic site for the xylanase domain (E280S), the ferulic acid esterase activity increased threefold, which suggests that the two catalytic domains for Xyn10D-Fae1A are functionally coupled. Directed mutagenesis of conserved residues for Xyl3A resulted in attenuation of activity, which supports the assignment of Xyl3A as a GH family 3 beta-D-xylosidase.

Crystal structure of fatty acid/phospholipid synthesis protein PlsX from Enterococcus faecalis

PlsX is a key enzyme that coordinates the production of fatty acids and membrane phospholipids. The plsX gene is co-localized with a bacterial fab gene cluster which encodes several key fatty acid biosynthetic enzymes. The protein is a member of a large, conserved protein family (Pfam02504) found exclusively in bacteria. The PlsX sequence homologues include both phosphate acetyltransferases and phosphate butaryltransferases that catalyze the transfer of an acetyl or butaryl group to orthophosphate. We have determined the crystal structure of PlsX from the human pathogen Enterococcus faecalis. PlsX is a alpha/beta/alpha sandwich that resembles a Rossmann fold and forms a dimer. A putative catalytic site has been identified within a deep groove on the interface between monomers. This site showed strong surface similarity to epimerases and reductases. It was recently proposed that PlsX is a phosphate acyltransferase that catalyzes the formation of acyl-phosphate from the acyl-acyl carrier protein; however the specific biochemical function of the PlsX protein awaits further experimental scrutiny.

Crystal structure of human retinoblastoma binding protein 9

Crystal structure of human retinoblastoma binding protein 9

Insights from the analysis of a predicted model of gp63 in Leishmania donovani

Leishmaniasis is a protozoal disease of human that occurs in most parts of the world. By considering the progress of bioinformatics in molecular modeling, major surface glycoprotein of Leishmania donovani (gp63) structure was modeled using homology modeling with high accuracy based on the X-ray crystal structure of the Leishmania major gp63 as a template, and then analyzed 3D structure of gp63 which can reveal exact facts about its structure and interaction. The objective of this study was to find folding and three dimensional structure of the gp63 as potent antigen for human. In this project, we applied the theory of evolution method, including comparative modeling and threading. This study presented a simple protocol for rapid and precise finding 3D structure of gp63 and investigation of its structural properties. The translated amino acid sequence showed that Leishmania donovani gp63 contains 590 amino acids precursor protein consisting of an NH(2)-terminal signal peptide of 39 amino acids for membrane targeting, a pro region of 48 amino acids, the mature protein of 478 amino acids containing glycosylation and putative catalytic sites, and a COOH-terminal signal peptide of 25 amino acids for GPI attachment. Based on our model, the protein consists of three domains: the N-terminal, central and C-terminal domains. Additionally, these results could guide future structure-function analyses of gp63 protein.

Defining the Topology of the N-Glycosylation Pathway in the Halophilic Archaeon Haloferax volcanii

In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to target proteins on the external surface of the cell. To remedy this situation, subcellular localization and topology predictive algorithms, protease accessibility, and immunoblotting, together with cysteine modification following site-directed mutagenesis, were enlisted to define the topology of Haloferax volcanii proteins experimentally proven to participate in the N-glycosylation process. AglJ and AglD, involved in the earliest and latest stages, respectively, of assembly of the pentasaccharide decorating the H. volcanii S-layer glycoprotein, were shown to present their soluble N-terminal domain, likely containing the putative catalytic site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A, and Mpg1-D, proteins putatively involved in this posttranslational event. The results thus point to the assembly of the pentasaccharide linked to certain Asn residues of the H. volcanii S-layer glycoprotein as occurring within the cell.

CENP-V is required for centromere organization, chromosome alignment and cytokinesis

The mechanism of mitotic chromosome condensation is poorly understood, but even less is known about the mechanism of formation of the primary constriction, or centromere. A proteomic analysis of mitotic chromosome scaffolds led to the identification of CENP-V, a novel kinetochore protein related to a bacterial enzyme that detoxifies formaldehyde, a by-product of histone demethylation in eukaryotic cells. Overexpression of CENP-V leads to hypercondensation of pericentromeric heterochromatin, a phenotype that is abolished by mutations in the putative catalytic site. CENP-V depletion in HeLa cells leads to abnormal expansion of the primary constriction of mitotic chromosomes, mislocalization and destabilization of the chromosomal passenger complex (CPC) and alterations in the distribution of H3K9me3 in interphase nucleoplasm. CENP-V-depleted cells suffer defects in chromosome alignment in metaphase, lagging chromosomes in anaphase, failure of cytokinesis and rapid cell death. CENP-V provides a novel link between centromeric chromatin, the primary constriction and the CPC.

A Novel Type of Thioredoxin Dedicated to Symbiosis in Legumes

Thioredoxins (Trxs) constitute a family of small proteins in plants. This family has been extensively characterized in Arabidopsis (Arabidopsis thaliana), which contains six different Trx types: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A detailed study of this family in the model legume Medicago truncatula, realized here, has established the existence of two isoforms that do not belong to any of the types previously described. As no possible orthologs were further found in either rice (Oryza sativa) or poplar (Populus spp.), these novel isoforms may be specific for legumes. Nevertheless, on the basis of protein sequence and gene structure, they are both related to Trxs m and probably have evolved from Trxs m after the divergence of the higher plant families. They have redox potential values similar to those of the classical Trxs, and one of them can act as a substrate for the M. truncatula NADP-Trx reductase A. However, they differ from classical Trxs in that they possess an atypical putative catalytic site and lack disulfide reductase activity with insulin. Another important feature is the presence in both proteins of an N-terminal extension containing a putative signal peptide that targets them to the endoplasmic reticulum, as demonstrated by their transient expression in fusion with the green fluorescent protein in M. truncatula or Nicotiana benthamiana leaves. According to their pattern of expression, these novel isoforms function specifically in symbiotic interactions in legumes. They were therefore given the name of Trxs s, s for symbiosis.

Functional and Structural Characterization of Four Glutaminases from Escherichia coli and Bacillus subtilis

Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.

The roles of Tyr91 and Lys162 in general acid–base catalysis in the pigeon NADP+-dependent malic enzyme

The role of general acid-base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr(91) and Lys(162) residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in k(cat) values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the k(cat) values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(k(cat)) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pK(a) value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys(162) as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr(91) residue could form a hydrogen bond with Lys(162) to act as a catalytic dyad that contributes a proton to complete the enol-keto tautomerization.

Modeling studies of potato nucleoside triphosphate diphosphohydrolase NTPDase1: an insight into the catalytic mechanism.

Nucleoside triphosphate diphosphohydrolase--NTPDase1 (apyrase, EC 3.6.1.5) was modeled based on sequence homology. The single polypeptide chain of apyrase is folded into two domains. The putative catalytic site with the apyrase conserved regions (ACR 1-5) is located between these two domains. Modeling confirmed that apyrase belongs to the actin superfamily of proteins. The amino acids interacting with the nucleoside triphosphate substrate and probably involved in the catalyzed hydrolysis were identified. The proposed two-step catalytic mechanism of hydrolysis involves Thr127 and Thr55 as potential nucleophilic factors responsible for the cleavage of the Pgamma and Pbeta anhydride bonds, respectively. Their action seems to be assisted by Glu170 and Glu78 residues, respectively. The presence of two nucleophiles in the active site of apyrase explains the differences in the hydrolytic activity between apyrases and other enzymes belonging to the NTPDase family.

Functional Coupling between the Kv1.1 Channel and Aldoketoreductase Kvβ1

The Shaker family voltage-dependent potassium channels (Kv1) assemble with cytosolic beta-subunits (Kvbeta) to form a stable complex. All Kvbeta subunits have a conserved core domain, which in one of them (Kvbeta2) is an aldoketoreductase that utilizes NADPH as a cofactor. In addition to this core, Kvbeta1 has an N terminus that closes the channel by the N-type inactivation mechanism. Point mutations in the putative catalytic site of Kvbeta1 alter the on-rate of inactivation. Whether the core of Kvbeta1 functions as an enzyme and whether its enzymatic activity affects N-type inactivation had not been explored. Here, we show that Kvbeta1 is a functional aldoketoreductase and that oxidation of the Kvbeta1-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP(+), induces a large increase in open channel current. The modulation is not affected by deletion of the distal C terminus of the channel, which has been suggested in structural studies to interact with Kvbeta. The rate of increase in current, which reflects NADPH oxidation, is approximately 2-fold faster at 0-mV membrane potential than at -100 mV. Thus, cofactor oxidation by Kvbeta1 is regulated by membrane potential, presumably via voltage-dependent structural changes in Kv1.1 channels.

Mind bomb 2, a founder myoblast-specific protein, regulates myoblast fusion and muscle stability

A fundamental step during Drosophila myogenesis is the specification of founder myoblasts (FMs). Founders possess the information required for the acquisition of muscle identity and for the execution of the myogenic programme, whereas fusion-competent myoblasts (FCMs) acquire this information after fusing to founders. Very little is known about genes that implement the execution of the myogenic programme. Here we characterise Mind bomb 2 (Mib2), a protein with putative E3 ubiquitin ligase activity that is exclusive of FMs and necessary for at least two distinct steps of the founder/myotube differentiation programme. Thus, in mib2 mutants, the early process of myoblast fusion is compromised, as FMs undergo a reduced number of rounds of fusion with FCMs. At later stages, with the onset of muscle contraction, many muscles degenerate, display aberrant sarcomeric structure and detach from tendons. The fusion process requires intact E3-RING-finger domains of Mib2 (the putative catalytic sites), probably to eliminate the FCM-specific activator Lmd from nascent myotubes. However, these sites appear dispensable for muscle integrity. This, and the subcellular accumulation of Mib2 in Z and M bands of sarcomeres, plus its physical interaction with nonmuscle myosin (a Z-band-localised protein necessary for the formation of myofibrils), suggest a structural role for Mib2 in maintaining sarcomeric stability. We suggest that Mib2 acts sequentially in myoblast fusion and sarcomeric stability by two separable processes involving distinct functions of Mib2.

How IRE1 Reacts to ER Stress

The long-awaited structure of the effector portion of IRE1, the endoplasmic reticulum stress transducer, is published in this issue of Cell (Lee et al., 2008). This structure provides new insight into the mysterious coupling of kinase and endoribonuclease activities in the oldest, most-conserved branch of the unfolded protein response in eukaryotes.

Structure of the Yeast tRNA m7G Methylation Complex

Loss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. In yeast, this modification is catalyzed by the heterodimeric complex composed of a catalytic subunit Trm8 and a noncatalytic subunit Trm82. We have solved the crystal structure of Trm8 alone and in complex with Trm82. Trm8 undergoes subtle conformational changes upon Trm82 binding which explains the requirement of Trm82 for activity. Cocrystallization with the S-adenosyl-methionine methyl donor defines the putative catalytic site and a guanine binding pocket. Small-angle X-ray scattering in solution of the Trm8-Trm82 heterodimer in complex with tRNA(Phe) has enabled us to propose a low-resolution structure of the ternary complex which defines the tRNA binding mode of Trm8-Trm82 and the structural elements contributing to specificity.

CLONING OF CHITINASE-LIKE PROTEIN1 CDNA FROM DICYEMID MESOZOANS (PHYLUM: DICYEMIDA)

Dicyemid mesozoans are endoparasites found in the renal sacs of benthic cephalopods. Adult dicyemids insert the distinct anterior region, termed a "calotte," into renal tubules of the host. We cloned cDNA encoding chitinase-like protein from the dicyemid Dicyema japonicum (Dicyema-clp 1), and also cloned the gene fragment corresponding to the cDNA. Dicyema-clp1 has the hydrophobic amino acid-rich region, but not the chitin-binding domains at the C terminus. Analyses using the SignalP prediction program suggest this hydrophobic amino acid-rich region is the anchor sequence to plasma membranes. The putative catalytic site in glyco18 domain exhibited 1 substitution from aspartic acid to asparagine. The gene fragment had short 9 introns (22-26 bp), and the coding sequence consisted of 10 exons (30-233 bp). Specific and strong expression of Dicyema-clpl was detected in the calotte of vermiform stages by whole mount in situ hybridization. N-acetyl-D-glucosamine was detected on the outer surface of both peripheral cells of dicyemids and epidermal cells of host renal appendages. Dicyema-clp appears to be associated with N-acetyl-D-glucosamine in the interface between dicyemid peripheral cells and epidermal cells of the host renal appendage, and possibly aids in adhering the calotte to host epidermal cells.

Different Mutations in the HHV-6 DNA Polymerase Gene Accounting for Resistance to Foscarnet

Human herpesvirus 6 (HHV-6), which is closely related to human cytomegalovirus, is sensitive to foscarnet (PFA). Up to now, the resistance of HHV-6 to PFA has not been investigated. The goal of the study was to isolate and characterize PFA-resistant HHV-6 mutants in order to determine the mechanisms of resistance to the drug. PFA-resistant viruses, isolated in MT4 cell culture under increasing concentrations of PFA, were characterized phenotypically and genotypically. The mutations identified in the HHV-6 DNA polymerase gene were evaluated in a functional assay using recombinant mutated forms of the enzyme, and their effect on protein structure was analysed in a three-dimensional model derived from available structures of DNA polymerases. Two mutants were selected and were 8- and 15-fold more resistant to PFA than the wild-type strain. Four amino acid changes were detected in the HHV-6 DNA polymerase in association with PFA resistance: T435R, H507Y, C525S, located in the deltaC conserved domain, and F292S. Either alone or in combination, these substitutions significantly decreased the inhibitory effect of PFA at the level of the polymerase, as measured by the incorporation of radiolabelled nucleotides in a DNA elongation assay. In the three-dimensional model of HHV-6 DNA polymerase structure the four changes were not located within the putative catalytic site, but they might induce either a disturbance of local conformation or a restricted access of PFA to its target site. This first characterization of HHV-6 resistance to PFA highlights the role of distinct DNA polymerase gene mutations.

Reconstitution of bluetongue virus polymerase activity from isolated domains based on a three‐dimensional structural model

Bluetongue virus (BTV) is a double‐stranded RNA virus of the Reoviridae family. The VP1 protein of BTV is the viral RNA‐dependent RNA polymerase (RdRp), which is responsible for the replication of the viral genome. Currently there is no structural information available for VP1. By manual alignment of BTV, Reovirus and other viral RdRps we have generated a model for the structure of VP1, the RdRp of BTV. The structure can be divided into three domains: an N‐terminal domain, a C‐terminal domain, and a central polymerase domain. Mutation of the putative catalytic site in the central polymerase domain by site‐directed mutagenesis abrogated in vitro replicase activity. Each of the domains was expressed individually and subsequently partially purified to obtain direct evidence for the location of polymerase activity and the nucleoside triphosphate binding site. The nucleoside triphosphate binding site was located by showing that CTP only bound to the full‐length protein or to the polymerase domain and not to either of the other two domains. None of the domains had catalytic activity when tested individually or in tandem but when all three domains were mixed together the RdRp activity was reconstituted. This is the first report of the reconstitution of a functional viral RdRp in vitro from individual domains. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 83–94, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

A phosphate‐binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 3'-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site.

Structure and expression of two genes encoding secreted acid phosphatases under phosphate-deficient conditions in Pholiota nameko strain N2

Twenty-three polypeptides secreted in response to a deficiency of inorganic phosphate (Pi) were previously found by two-dimensional polyacrylamide gel electrophoresis analysis in mycelia of Pholiota nameko strain N2. In this study, N-terminal sequencing revealed three of them to be identical to known acid phosphatases of P. nameko strain N114. Two cDNAs and the corresponding genomic DNAs of genes PNAP1 and PNAP2 which encode two of the three acid phosphatases were cloned. The deduced amino acid sequences of PNAP1 and PNAP2 showed high similarity to other fungal acid phosphatases and contained a putative catalytic active site of acid phosphatase. PNAP1 and PNAP2 are comprised of five and seven exons interrupted by four and six introns, respectively. Their promoter regions include two cis-acting elements found in Pi deficiency-inducible genes of Saccharomyces cerevisiae, together with several known functional elements such as a TATA box. Northern blot analysis showed that PNAP1 and PNAP2 are expressed in response to a deficiency of Pi.