Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0348321 (Haemophilus)
 15,372 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cellular redox control is often mediated by oxidation and reduction of cysteine residues in the redox-sensitive proteins, where thioredoxin and glutaredoxin (Grx) play as electron donors for the oxidized proteins. Despite the importance of protein-protein interactions between the electron donor and acceptor proteins, there has been no structural information for the interaction of thioredoxin or Grx with natural target proteins. Here, we present the crystal structure of a novel Haemophilus influenza peroxiredoxin (Prx) hybrid Prx5 determined at 2.8-A resolution. The structure reveals that hybrid Prx5 forms a tightly associated tetramer where active sites of Prx and Grx domains of different monomers interact with each other. The Prx-Grx interface comprises specific charge interactions surrounded by weak interactions, providing insight into the target recognition mechanism of Grx. The tetrameric structure also exhibits a flexible active site and alternative Prx-Grx interactions, which appear to facilitate the electron transfer from Grx to Prx domain. Differences of electron donor binding surfaces in Prx proteins revealed by an analysis based on the structural information explain the electron donor specificities of various Prx proteins.
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PMID:The tetrameric structure of Haemophilus influenza hybrid Prx5 reveals interactions between electron donor and acceptor proteins. 1252 27

Since they are equipped with several strategies by which they evade the antimicrobial defense of host macrophages, it is surprising that members of the genus Haemophilus appear to be deficient in common antioxidant systems that are well established to protect prokaryotes against oxidative stress. Among others, no genetic evidence for glutathione (gamma-Glu-Cys-Gly) (GSH) biosynthesis or for alkyl hydroperoxide reduction (e.g., the Ahp system characteristic or enteric bacteria) is apparent from the Haemophilus influenzae Rd genome sequence, suggesting that the organism relies on alternative systems to maintain redox homeostasis or to reduce small alkyl hydroperoxides. In this report we address this apparent paradox for the nontypeable H. influenzae type strain NCTC 8143. Instead of biosynthesis, we could show that this strain acquires GSH by importing the thiol tripeptide from the growth medium. Although such GSH accumulation had no effect on growth rates, the presence of cellular GSH protected against methylglyoxal, tert-butyl hydroperoxide (t-BuOOH), and S-nitrosoglutathione toxicity and regulated the activity of certain antioxidant enzymes. H. influenzae NCTC 8143 extracts were shown to contain GSH-dependent peroxidase activity with t-BuOOH as the peroxide substrate. The GSH-mediated protection against t-BuOOH stress is most probably catalyzed by the product of open reading frame HI0572 (Prx/Grx), which we isolated from a genomic DNA fragment that confers wild-type resistance to t-BuOOH toxicity in the Ahp-negative Escherichia coli strain TA4315 and that introduces GSH-dependent alkyl hydroperoxide reductase activity into naturally GSH peroxidase-negative E. coli. Finally, we demonstrated that cysteine is an essential amino acid for growth and that cystine, GSH, glutathione amide, and cysteinylglycine can be catabolized in order to complement cysteine deficiency.
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PMID:Exogenous glutathione completes the defense against oxidative stress in Haemophilus influenzae. 1259 74

While belonging to the same family of antioxidant enzymes, members of the peroxiredoxins do not necessarily employ one and the same method for their reduction. Most representatives become reduced with the aid of thioredoxin, whereas some members use AhpF, tryparedoxin, or cyclophilin A. Recent research on a new peroxiredoxin isoform (type C) from Populus trichocarpa has shown that these particular types may also use glutaredoxin instead of thioredoxin. This finding is supported by the occurrence of chimeric proteins composed of a peroxiredoxin and glutaredoxin region. A gene encoding such a fusion protein is enclosed in the Haemophilus influenzae Rd genome. We expressed the H. influenzae protein, denoted here as PGdx, in Escherichia coli and purified the recombinant enzyme. In vitro assays demonstrate that PGdx, in the presence of dithiothreitol or glutathione, is able to protect supercoiled DNA against the metal ion-catalyzed oxidation-system. Enzymatic assays did, indeed, characterize PGdx as a peroxidase, requiring the glutathione redox cycle for the reduction of hydrogen peroxide (k(cat)/K(m) 5.01 x 10(6) s(-1) m(-1)) as well as the small organic hydroperoxide tert-butylhydroperoxide (k(cat)/K(m) 5.67 x 10(4) s(-1) m(-1)). Enzymatic activity as function of the glutathione concentration deviated from normal Michaelis-Menten kinetics, giving a sigmoidal pattern with an apparent Hill coefficient of 2.9. Besides the formation of a disulfide-linked PGdx dimer, it was also shown by mass spectrometric analysis that cysteine 49, which is equivalent to the active site cysteine of the peroxiredoxins, undergoes glutathionylation during purification under nonreducing conditions. Based on these results, we propose a model for the catalytic mechanism.
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PMID:Purification and characterization of a chimeric enzyme from Haemophilus influenzae Rd that exhibits glutathione-dependent peroxidase activity. 1260 54

The olpA gene of Chryseobacterium meningosepticum, encoding a molecular class C phosphatase, was cloned and expressed in Escherichia coli. The gene encodes a 29-kDa polypeptide containing an amino-terminal signal peptide typical of bacterial membrane lipoproteins. Expression in E. coli results in a functional product that mostly partitions in the outer membrane. A secreted soluble OlpA derivative (sOlpA) lacking the N-terminal cysteine residue for lipid anchoring was produced in E. coli and purified by means of two steps of ion exchange chromatography. Analysis of the kinetic parameters of sOlpA with several organic phosphoesters revealed that the enzyme was able to efficiently hydrolyze nucleotide monophosphates, with a strong preference for 5'-nucleotides and for 3'-AMP. The enzyme was also able to hydrolyze sugar phosphates and beta-glycerol phosphate, although with a lower efficiency, whereas it was apparently inactive against nucleotide di- and triphosphates, diesters, and phytate. OlpA, therefore, can be considered a broad-spectrum nucleotidase with preference for 5'-nucleotides. Its functional behaviour exhibits differences from that of the Haemophilus influenzae OMP P4 lipoprotein, revealing functional heterogeneity among phosphatases of molecular class C.
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PMID:The molecular class C acid phosphatase of Chryseobacterium meningosepticum (OlpA) is a broad-spectrum nucleotidase with preferential activity on 5'-nucleotides. 1275 63

Serine acetyltransferase (SAT, EC 2.3.1.30) catalyzes the CoA-dependent acetylation of the side chain hydroxyl group of l-serine to form O-acetylserine, as the first step of a two-step biosynthetic pathway in bacteria and plants leading to the formation of l-cysteine. This reaction represents a key metabolic point of regulation for the cysteine biosynthetic pathway due to its feedback inhibition by cysteine. We have determined the X-ray crystal structure of Haemophilus influenzae SAT in complexes with CoA and its cysteine feedback inhibitor. The enzyme is a 175 kDa hexamer displaying the characteristic left-handed parallel beta-helix (LbetaH) structural domain of the hexapeptide acyltransferase superfamily of enzymes. Cysteine is bound in a crevice between adjacent LbetaH domains and underneath a loop excluded from the coiled LbetaH. The proximity of its thiol group to the thiol group of CoA derived from superimposed models of the cysteine and CoA complexes confirms that cysteine is bound at the active site. Analysis of the contacts of SAT with cysteine and CoA and the conformational differences that distinguish these complexes provides a structural basis for cysteine feedback inhibition, which invokes competition between cysteine and serine binding and a cysteine-induced conformational change of the C-terminal segment of the enzyme that excludes binding of the cofactor.
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PMID:Structure of serine acetyltransferase in complexes with CoA and its cysteine feedback inhibitor. 1514 85

One component of the anti-microbial function of lactoferrin (Lf) is its ability to sequester iron from potential pathogens. To overcome this iron limitation, a number of gram-negative bacterial pathogens have developed a mechanism for acquiring iron directly from this host glycoprotein. This mechanism involves surface receptors capable of specifically binding Lf from the host, removing iron and transporting it across the outer membrane. The iron is then bound by a periplasmic iron-binding protein, FbpA, and transported into the cell via an inner membrane complex comprised of FbpB and FbpC. The receptor has been shown to consist of two proteins, LbpA and LbpB. LbpB is bilobed lipoprotein anchored to the outer membrane via fatty acyl groups attached to the N-terminal cysteine. LbpA is a homologue of siderophore receptors, which consist of an N-terminal plug and a C-terminal beta-barrel region. We propose that the receptor proteins, LbpA and LbpB, induce conformational changes in human Lf (hLf) that lower its affinity for iron that binding by FbpA can drive the transport across the outer membrane, a mechanism shared with transferrin (Tf) receptors. The interaction between the receptor proteins and Lf is quite extensive and has been previously studied by using chimeric proteins comprised of Lf & Tf. In an attempt to evaluate the role of FbpA in the transport process, a series of site-directed mutants of FbpA were prepared and used to replace the wild-type protein in the iron acquisition pathway. The mutations were made in the iron-binding and anion-binding ligands of FbpA and were designed to result in altered binding properties. Protein crystallography of the iron-bound form of the Q58L mutant protein revealed that it was in the open conformation with iron coordinated by Y195 and Y196 from the C-terminal domain but not by the other iron-liganding amino acids from the N-terminal domain, H9 and E57. Replacement of the native FbpA in Neisseria meningitidis with wild-type or mutant Haemophilus influenzae FbpAs resulted in a defect in growth on Tf or Lf, suggesting that there may be a barrier to functional expression of H. influenzae FbpAs in Neisseria meningitidis. Thus mutants of the N. meningitidis FbpA are being prepared to replace wild-type protein in order to test their ability to mediate transport from hLf.
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PMID:Lactoferrin receptors in gram-negative bacteria: insights into the iron acquisition process. 1522 71

The kinetic mechanism of serine acetyltransferase from Haemophilus influenzae was studied in both reaction directions. The enzyme catalyzes the conversion of acetyl CoA and L-serine to O-acetyl-L-serine (OAS) and coenzyme A (CoASH). In the direction of L-serine acetylation, an equilibrium ordered mechanism is assigned at pH 6.5. The initial velocity pattern in the absence of added inhibitors is best described by a series of lines converging on the ordinate when L-serine is varied at different fixed levels of acetyl CoA. The initial velocity pattern at pH 7.5 is also intersecting, but the lines are nearly parallel. Product inhibition by OAS is noncompetitive against acetyl CoA, while it is uncompetitive against L-serine. Product inhibition by L-serine in the reverse reaction direction is noncompetitive with respect to both OAS and CoASH. Glycine and S-methyl-L-cysteine (SMC) were used as dead-end analogs of L-serine and OAS, respectively. Glycine is competitive versus L-serine and uncompetitive versus acetyl CoA, while SMC is competitive against OAS and uncompetitive against CoASH. Desulfo-CoA was used as a dead-end analog of both acetyl CoA and CoASH, and is competitive versus both substrates in the direction of L-serine acetylation; while it is competitive against CoASH and noncompetitive against OAS in the direction of CoASH acetylation. All of the above kinetic parameters are consistent with those predicted for an ordered mechanism at pH 6.5 with the exception of the uncompetitive inhibition by OAS vs. serine. The latter inhibition pattern suggests combination of OAS with the central E:acetyl CoA:serine complex. Cysteine is known to regulate its own biosynthesis at the level of SAT. As a dead-end inhibitor, L-cysteine is competitive against both substrates in both reaction directions. These results are discussed in terms of the mechanism of regulation.
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PMID:Kinetic mechanism of the serine acetyltransferase from Haemophilus influenzae. 1531 14

Prolyl-tRNA synthetases (ProRSs) from all three domains of life have been shown to misactivate cysteine and to mischarge cysteine onto tRNAPro. Although most bacterial ProRSs possess an amino acid editing domain that deacylates mischarged Ala-tRNAPro, editing of Cys-tRNAPro has not been demonstrated and a double-sieve mechanism of editing does not appear to be sufficient to eliminate all misacylated tRNAPro species from the cell. It was recently shown that a ProRS paralog, the YbaK protein from Haemophilus influenzae, which is homologous to the ProRS editing domain, is capable of weakly deacylating Ala-tRNAPro. This function appears to be redundant with that of its corresponding ProRS, which contains a canonical bacterial editing domain. In the present study, we test the specificity of editing by H. influenzae YbaK and show that it efficiently edits Cys-tRNAPro and that a conserved Lys residue is essential for this activity. These findings represent the first example of an editing domain paralog possessing altered specificity and suggest that similar autonomous editing domains could act upon different mischarged tRNAs thus providing cells with enhanced proofreading potential. This work also suggests a novel mechanism of editing wherein a third sieve is used to clear Cys-tRNAPro in at least some organisms.
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PMID:Trans-editing of Cys-tRNAPro by Haemophilus influenzae YbaK protein. 1532 38

Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes the reductive dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde in the aspartate biosynthetic pathway. This pathway is not found in humans or other eukaryotic organisms, yet is required for the production of threonine, isoleucine, methionine and lysine in most microorganisms. The mechanism of this enzyme has been examined through the structures of two active-site mutants of ASADH from Haemophilus influenzae. Replacement of the enzyme active-site cysteine with serine (C136S) leads to a dramatic loss of catalytic activity caused by the expected decrease in nucleophilicity, but also by a change in the orientation of the serine hydroxyl group relative to the cysteine thiolate. In contrast, in the H277N active-site mutant the introduced amide is oriented in virtually the same position as that of the histidine imidazole ring. However, a shift in the position of the bound reaction intermediate to accommodate this shorter asparagine side chain, coupled with the inability of this introduced amide to serve as a proton acceptor, results in a 100-fold decrease in the catalytic efficiency of H277N relative to the native enzyme. These mutant enzymes have the same overall fold and high structural identity to native ASADH. However, small perturbations in the positioning of essential catalytic groups or reactive intermediates have dramatic effects on catalytic efficiency.
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PMID:Critical catalytic functional groups in the mechanism of aspartate-beta-semialdehyde dehydrogenase. 1538 27

IscU is a highly conserved protein that serves as the scaffold for IscS-mediated assembly of iron-sulfur ([Fe-S]) clusters. We report the NMR solution structure of monomeric Haemophilus influenzae IscU with zinc bound at the [Fe-S] cluster assembly site. The compact core of the globular structure has an alpha-beta sandwich architecture with a three-stranded antiparallel beta-sheet and four alpha-helices. A nascent helix is located N-terminal to the core structure. The zinc is ligated by three cysteine residues and one histidine residue that are located in and near conformationally dynamic loops at one end of the IscU structure. Removal of the zinc metal by chelation results in widespread loss of structure in the apo form. The zinc-bound IscU may be a good model for iron-loaded IscU and may demonstrate structural features found in the [Fe-S] cluster bound form. Structural and functional similarities, genomic context in operons containing other homologous genes, and distributions of conserved surface residues support the hypothesis that IscU protein domains are homologous (i.e. derived from a common ancestor) with the SufE/YgdK family of [Fe-S] cluster assembly proteins.
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PMID:Solution NMR structure of the iron-sulfur cluster assembly protein U (IscU) with zinc bound at the active site. 1552 5


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