Malonyl-CoA: acyl carrier protein transacylase from Helicobacter pylori: Crystal structure and its interaction with acyl carrier protein

Malonyl-CoA: acyl carrier protein transacylase (MCAT) is a critical enzyme responsible for the transfer of the malonyl moiety to holo-acyl carrier protein (ACP) forming the malonyl-ACP intermediates in the initiation step of type II fatty acid synthesis (FAS II) in bacteria. MCAT has been considered as an attractive drug target in the discovery of antibacterial agents. In this study, the crystal structure of MCAT from Helicobacter pylori (Hp) at 2.5 angstroms resolution is reported, and the interaction of HpMCAT with HpACP is extensively investigated by using computational docking, GST-pull-down, and surface plasmon resonance (SPR) technology-based assays. The crystal structure results reveal that HpMCAT has a compact folding composed of a large subdomain with a similar core as in alpha/beta hydrolases, and a similar ferredoxin-like small subdomain as in acylphosphatases. The docking result suggests two positively charged areas near the entrance of the active site of HpMCAT as the ACP-binding region. Binding assay research shows that HpMCAT demonstrates a moderately binding ability against HpACP. The solved 3D structure of HpMCAT is expected to supply useful information for the structure-based discovery of novel inhibitors against MCAT, and the quantitative study of HpMCAT interaction with HpACP is hoped to give helpful hints in the understanding of the detailed catalytic mechanisms for HpMCAT.     

Crystal structure of enoyl-acyl carrier protein reductase (FabK) from Streptococcus pneumoniae reveals the binding mode of an inhibitor

Enoyl-acyl carrier protein (ACP) reductases are critical for bacterial type II fatty acid biosynthesis and thus are attractive targets for developing novel antibiotics. We determined the crystal structure of enoyl-ACP reductase (FabK) from Streptococcus pneumoniae at 1.7 A resolution. There was one dimer per asymmetric unit. Each subunit formed a triose phosphate isomerase (TIM) barrel structure, and flavin mononucleotide (FMN) was bound as a cofactor in the active site. The overall structure was similar to the enoyl-ACP reductase (ER) of fungal fatty acid synthase and to 2-nitropropane dioxygenase (2-ND) from Pseudomonas aeruginosa, although there were some differences among these structures. We determined the crystal structure of FabK in complex with a phenylimidazole derivative inhibitor to envision the binding site interactions. The crystal structure reveals that the inhibitor binds to a hydrophobic pocket in the active site of FabK, and this is accompanied by induced-fit movements of two loop regions. The thiazole ring and part of the ureido moiety of the inhibitor are involved in a face-to-face pi-pi stacking interaction with the isoalloxazine ring of FMN. The side-chain conformation of the proposed catalytic residue, His144, changes upon complex formation. Lineweaver-Burk plots indicate that the inhibitor binds competitively with respect to NADH, and uncompetitively with respect to crotonoyl coenzyme A. We propose that the primary basis of the inhibitory activity is competition with NADH for binding to FabK, which is the first step of the two-step ping-pong catalytic mechanism.     

Molecular basis for triclosan activity involves a flipping loop in the active site

The crystal structure of the Escherichia coli enoyl reductase-NAD+-triclosan complex has been determined at 2.5 A resolution. The Ile192-Ser198 loop is either disordered or in an open conformation in the previously reported structures of the enzyme. This loop adopts a closed conformation in our structure, forming van der Waals interactions with the inhibitor and hydrogen bonds with the bound NAD+ cofactor. The opening and closing of this flipping loop is likely an important factor in substrate or ligand recognition. The closed conformation of the loop appears to be a critical feature for the enhanced binding potency of triclosan, and a key component in future structure-based inhibitor design.     

Crystal structure of the secreted protein HP1454 from the human pathogen Helicobacter pylori

HP1454 is a protein of 303 amino acids found in the extracellular milieu of Helicobacter pylori. The protein structure, crystallized in the orthorhombic C222₁ space group with one molecule per asymmetric unit, has been determined using the single‐wavelength anomalous dispersion method. HP1454 exhibits an elongated bent shape, composed of three distinct domains. Each domain possesses a fold already present in other structures: Domain I contains a three‐strand antiparallel β‐barrel flanked by a long α‐helix, Domain II is an anti‐parallel three‐helix bundle, and Domain III a β‐sheet flanked by two α‐helices. The overall assembly of the protein does not bear any similarity with known structures. Proteins 2014; 82:2868–2873. © 2014 Wiley Periodicals, Inc.     

cDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli

The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastidlocalized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.     

Purification and characterization of protein methylase II from Helicobacter pylori

Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS-PAGE. On non-denaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4x(78+29)=428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a K(m) value of 5.0x10(-6) M for S-adenosyl-L-methionine and a V(max) of 205 pmol methyl-(14)C transferred min(-1) mg(-1) protein.     

Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori

D-Alanine-D-alanine ligase is the second enzyme in the D-Ala branch of bacterial cell wall peptidoglycan assembly, and recognized as an attractive antimicrobial target. In this work, the D-Ala-D-Ala ligase of Helicobacter pylori strain SS1 (HpDdl) was kinetically and structurally characterized. The determined apparent K(m) of ATP (0.87 microM), the K(m1) (1.89 mM) and K(m2) of D-Ala (627 mM), and the k(cat) (115 min(-1)) at pH 8.0 indicated its relatively weak binding affinity and poor catalytic activity against the substrate D-Ala in vitro. However, by complementary assay of expressing HpDdl in Escherichia coli Delta ddl mutant, HpDdl was confirmed to be capable of D-Ala-D-Ala ligating in vivo. Through sequence alignment with other members of the D-Ala-D-X ligase superfamily, HpDdl keeps two conservatively substituted residues (Ile16 and Leu241) and two nonconserved residues (Leu308 and Tyr311) broadly located in the active region of the enzyme. Kinetic analyses against the corresponding HpDdl mutants (I16V, L241Y, L241F, L308T, and Y311S) suggested that these residues, especially Leu308 and Tyr311, might partly contribute to the unique catalytic properties of the enzyme. This was fairly proved by the crystal structure of HpDdl, which revealed that there is a 3(10)-helix (including residues from Gly306 to Leu312) near the D-Ala binding region in the C-terminal domain, where HpDdl has two sequence deletions compared with other homologs. Such 3(10)-helix may participate in D-Ala binding and conformational change of the enzyme. Our present work hopefully provides useful information for understanding the D-Ala-D-Ala ligase of Helicobacter pylori.     

Crystal structure of Helicobacter pylori spermidine synthase: a Rossmann-like fold with a distinct active site

Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine during spermidine biosynthesis. Helicobacter pylori PAPT (HpPAPT) has a low sequence identity with other PAPTs and lacks the signature sequence found in other PAPTs. The crystal structure of HpPAPT, determined by multiwavelength anomalous dispersion, revealed an N-terminal beta-stranded domain and a C-terminal Rossmann-like domain. Structural comparison with other PAPTs showed that HpPAPT has a unique binding pocket between two domains, numerous non-conserved residues, a less acidic electrostatic surface potential, and a large buried space within the structure. HpPAPT lacks the gatekeeping loop that facilitates substrate binding in other PAPTs. PAPTs are essential for bacterial cell viability; thus, HpPAPT may be a potential antimicrobial drug target for H. pylori owing to its characteristic PAPT sequence and distinct conformation.     

Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1

Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and K_m values of the two substrates dUMP and CH₂H₄folate were determined to be 15.3 ± 1.25 μM and 0.35 ± 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti‐H. pylori inhibitor discovery.     

Activities of muscadine grape skin and quercetin against Helicobacter pylori infection in mice

Aims: To explore the preventative potential of muscadine grape skin (MGS) and the single flavonoid, quercetin, as an alternative means for ameliorating Helicobacter pylori infection and/or the H. pylori‐induced inflammatory response in mice. Methods and Results: The antimicrobial and anti‐inflammatory properties of MGS and quercetin, a major phenolic constituent, were evaluated against H. pylori in vitro and in vivo. The antimicrobial activity of quercetin was evaluated against 11 H. pylori strains in vitro with inhibition of all strains at 128–64 μg ml^?1. In vivo studies showed a moderate reduction in H. pylori counts following treatment with 5 and 10% MGS or quercetin (25 mg kg^?1 body weight) in addition to significantly reduced inflammatory cytokines (TNF‐α, IL‐1β and IFN‐γ) when compared with untreated mice. Conclusions: MGS and quercetin did not significantly reduce H. pylori growth in a mouse model. However, these products were effective in regulating the inflammatory response to H. pylori infection. Significance and Impact of the Study: Our results suggest that H. pylori infection may be reduced or prevented via the consumption of fruits rich in certain phenolic compounds (e.g. quercetin) such as muscadine grapes.     

The role of interleukin-17 in the Helicobacter pylori induced infection and immunity

Background: Helicobacter pylori infection is regarded as the major cause of various gastric diseases and induces the production of several cytokines including interleukin‐17 (IL‐17) recently recognized as an important player in the mammalian immune system. Objective: This review deals with the role of IL‐17 on the H. pylori‐induced infection and immunity in humans and experimental animals. Results: H. pylori infection increases IL‐17 in the gastric mucosa of humans and experimental animals. In humans, IL‐17 induces the secretion of IL‐8 by activating the ERK 1/2 MAP kinase pathway and the released IL‐8 attracts neutrophils promoting inflammation. IL‐23 is increased in patients with H. pylori‐related gastritis and regulates IL‐17 secretion via STAT3 pathway. Studies in H. pylori‐infected mice indicate that IL‐17 is primarily associated with gastric inflammation. The early events in the immune response of immunized and challenged mice include the recruitment of T cells and the production of IL‐17. Neutrophil attracting chemokines are released, and the bacterial load is considerably reduced. IL‐17 plays a dual role in infection and vaccination. In infection, T regulatory cells (Tregs) suppress the inflammatory reaction driven by IL‐17 thereby favoring bacterial persistence. Immunization produces Helicobacter‐specific memory T‐helper cells that can possibly alter the ratio between T‐helper 17 and Treg responses so that the IL‐17‐driven inflammatory reaction can overcome the Treg response leading to bacterial clearance. Conclusion: IL‐17 plays an important role in H. pylori‐related gastritis and in the reduction of Helicobacter infection in mice following immunization.     

Functional analysis of the roles of FliQ and FlhB in flagellar expression in Helicobacter pylori

Expression of the two Helicobacter pylori flagellin proteins FlaA and FlaB is required for full motility and persistent infection of the gastric mucosa. The mechanisms and regulation of the biosynthesis and export of flagella in H. pylori are still poorly understood. Scrutiny of the H. pylori 26695 genome sequence revealed homologues of FliQ and FlhB. The roles of the fliQ and flhB genes in H. pylori were investigated by the construction and characterisation of defined isogenic mutants. The results indicate that these genes are involved in the flagellar expression, adhesion to and colonisation of the gastric mucosa.     

Characterization and inhibitor discovery of one novel malonyl-CoA: acyl carrier protein transacylase (MCAT) from Helicobacter pylori

Type II fatty acid synthesis (FAS II) is an essential process for bacteria survival, and malonyl-CoA:acyl carrier protein transacylase (MCAT) is a key enzyme in FAS II pathway, which is responsible for transferring the malonyl group from malonyl-CoA to the holo-ACP by forming malonyl-ACP. In this work, we described the cloning, characterization and enzymatic inhibition of a new MCAT from Helicobacter pylori strain SS1 (HpMCAT), and the gene sequence of HpfabD was deposited in the GenBank database (Accession No. AY738332 ). Enzymatic characterization of HpMCAT showed that the K(m) value for malonyl-CoA was 21.01+/-2.3 microM, and the thermal- and guanidinium hydrochloride-induced unfolding processes for HpMCAT were quantitatively investigated by circular dichroism spectral analyses. Moreover, a natural product, corytuberine, was discovered to demonstrate inhibitory activity against HpMCAT with IC(50) value at 33.1+/-3.29 microM. Further enzymatic assay results indicated that corytuberine inhibits HpMCAT in an uncompetitive manner. To our knowledge, this is the firstly reported MCAT inhibitor to date. This current work is hoped to supply useful information for better understanding the MCAT features of H. pylori strain, and corytuberine might be used as a potential lead compound in the discovery of the antibacterial agents using HpMCAT as target.     

Mutations of Helicobacter pylori associated with fluoroquinolone resistance in Korea

Background and Aim: Fluoroquinolone resistance of Helicobacter pylori is known to be dependent on mutations in the QRDR of gyrA. This study was performed to investigate the distribution of gyrA point mutations and to evaluate the impact of the mutations on second‐line H. pylori eradication therapy. Methods: After H. pylori isolation from gastric mucosal specimens, fluoroquinolone resistance was examined using the agar dilution method. DNA sequencing of the QRDR of gyrA was performed in 89 fluoroquinolone‐resistant and 27 fluoroquinolone‐susceptible isolates. Transformation experiments were performed to confirm mutations in the resistant strains. The eradication rates of moxifloxacin‐containing triple therapy were evaluated depending on the resistance of fluoroquinolone. Results: The gyrA mutations were detected in 75.3% (55 of 73 strains) of the primary resistant strains and 100% (16 strains) of the secondary resistant strains. The most common mutations were Asp‐91 (36.0%) and Asn‐87 (33.7%). The MIC values in the transformed strains differed depending on the gyrA mutations, N87, and D91. Six patients with fluoroquinolone‐resistant strains received moxifloxacin‐containing triple therapy as the second‐line therapy, and two of three patients with Asn‐87 mutations (66.7%) failed in the eradication. By contrast, three patients with Asp‐91 mutations had successful eradication treatment. Conclusions: Fluoroquinolone resistance of H. pylori was caused by gyrA Asn‐87 and Asp‐91 point mutations. The Asn‐87 mutation seems to be an important determinant of failure of fluoroquinolone‐containing triple eradication therapy based on eradication results.     

Crystal structure of a substrate complex of Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III (FabH) with lauroyl-coenzyme A

Beta-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes a two step reaction that initiates the pathway of fatty acid biosynthesis in plants and bacteria. In Mycobacterium tuberculosis, FabH catalyzes extension of lauroyl, myristoyl and palmitoyl groups from which cell wall mycolic acids of the bacterium are formed. The first step of the reaction is an acyl group transfer from acyl-coenzyme A to the active-site cysteine of the enzyme; the second step is acyl chain extension by two carbon atoms through Claisen condensation with malonyl-acyl carrier protein. We have previously determined the crystal structure of a type II, dissociated M.tuberculosis FabH, which catalyzes extension of lauroyl, myristoyl and palmitoyl groups. Here we describe the first long-chain Michaelis substrate complex of a FabH, that of lauroyl-coenzyme A with a catalytically disabled Cys-->Ala mutant of M.tuberculosis FabH. An elongated channel extending from the mutated active-site cysteine defines the acyl group binding locus that confers unique acyl substrate specificity on M.tuberculosis FabH. CoA lies in a second channel, bound primarily through interactions of its nucleotide group at the enzyme surface. The apparent weak association of CoA in this complex may play a role in the binding and dissociation of long chain acyl-CoA substrates and products and poses questions pertinent to the mechanism of this enzyme.