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)

A branched-chain aminotransferase gene (ilvE) from Lactococcus lactis LM0230 was identified on a 9-kb chromosomal insert by complementation in Escherichia coli DL39. Sequencing of a 2.0-kbp fragment resulted in the identification of a 1,023-bp open reading frame that could encode a 340-amino-acid protein. Sequence analysis of the deduced amino acid sequence revealed 62% identity to IlvE of Haemophilus influenzae and high similarity to IlvEs from a variety of organisms found in GenBank classified as class IV aminotransferases. Under logarithmic growth in complex medium, ilvE is transcribed monocistronically as a 1.1-kb transcript. Hydrophobicity plot analysis of the deduced amino acid sequence and the lack of a signal peptide sequence suggest IlvE is a cytosolic protein. A derivative of LM0230 lacking IlvE activity was constructed by gene replacement. Comparison of the IlvE-deficient strain's ability to grow in defined media lacking an amino acid but containing its alpha-keto acid biosynthetic precursor to that of the wild-type strain indicated that IlvE is the only enzyme capable of synthesis of Ile and Val from their biosynthetic precursors. Comparison of the aminotransferase activity of the IlvE mutant to LM0230 revealed that the mutant retained <2, 4.5, 43, 40, and 76% of its aminotransferase activity with Ile, Val, Leu, Met, and Phe, respectively. No difference in growth or acidification rate between LM0230 and the IlvE-deficient strain was observed in milk.
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PMID:Gene cloning, sequencing, and inactivation of the branched-chain aminotransferase of Lactococcus lactis LM0230. 1083 6

The first unique step in bacterial and plant methionine biosynthesis involves the acylation of the gamma-hydroxyl of homoserine. In Haemophilus influenzae, acylation is accomplished via an acetyl-CoA-dependent acetylation catalyzed by homoserine transacetylase. The activity of this enzyme regulates flux of homoserine into multiple biosynthetic pathways and, therefore, represents a critical control point for cell growth and viability. We have cloned homoserine transacetylase from H. influenzae and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the acetyl group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-acetylhomoserine. The maximal velocity and V/K(homoserine) were independent of pH over the range of values tested, while V/K(acetyl)(-)(CoA) was dependent upon the ionization state of a single group exhibiting a pK value of 8.6, which was required to be protonated. Solvent kinetic isotope effect studies yielded inverse effects of 0.75 on V and 0.74 on V/K(CoA) on the reverse reaction and effects of 1.2 on V and 1.7 on V/K(homoserine) on the forward reaction. Direct evidence for the formation of an acetyl-enzyme intermediate was obtained using rapid-quench labeling studies. On the basis of these observations, we propose a chemical mechanism for this important member of the acyltransferase family and contrast its mechanism with that of homoserine transsuccinylase.
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PMID:Enzyme-catalyzed acylation of homoserine: mechanistic characterization of the Haemophilus influenzae met2-encoded homoserine transacetylase. 1091 62

New inhibitors of peptide deformylase (PDF) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. Several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) The inhibition of Escherichia coli growth could be counteracted by overexpression of PDF from different organisms, including E. coli, Streptococcus pneumoniae, and Haemophilus influenzae. Conversely, reduced expression of PDF in S. pneumoniae resulted in an increased susceptibility to the inhibitors. (ii) Proteome analysis on two-dimensional gels revealed a shift for many proteins towards lower pI in the presence of PDF inhibitors, as would be expected if the proteins still carry their N-formyl-Met terminus. (iii) PDF inhibitors show no antimicrobial activity against E. coli under conditions that make growth independent of formylation and deformylation. The antibacterial activity in E. coli was characterized as bacteriostatic. Furthermore, the development of resistance in E. coli was observed to occur with high frequency (10(-7)). Resistant mutants show a reduced growth rate, and DNA sequence analysis revealed mutations in their formyl transferase gene. Taking all these aspects into account, we conclude that PDF may not be an optimal target for broad-spectrum antibacterial agents.
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PMID:Peptide deformylase as an antibacterial drug target: target validation and resistance development. 1125 16

The affinity of [(3)H]benzylpenicillin for penicillin-binding protein (PBP) 3A was reduced in 25 clinical isolates of beta-lactamase-negative ampicillin (AMP)-resistant (BLNAR) Haemophilus influenzae for which the AMP MIC was > or =1.0 microg/ml. The affinities of PBP 3B and PBP 4 were also reduced in some strains. The sequences of the ftsI gene encoding the transpeptidase domain of PBP 3A and/or PBP 3B and of the dacB gene encoding PBP 4 were determined for these strains and compared to those of AMP-susceptible Rd strains. The BLNAR strains were classified into three groups on the basis of deduced amino acid substitutions in the ftsI gene, which is thought to be involved in septal peptidoglycan synthesis. His-517, near the conserved Lys-Thr-Gly (KTG) motif, was substituted for Arg-517 in group I strains (n = 9), and Lys-526 was substituted for Asn-526 in group II strains (n = 12). In group III strains (n = 4), three residues (Met-377, Ser-385, and Leu-389), positioned near the conserved Ser-Ser-Asn (SSN) motif, were replaced with Ile, Thr, and Phe, respectively, in addition to the replacement with Lys-526. The MICs of cephem antibiotics with relatively high affinities for PBP 3A and PBP 3B were higher than those of AMP and meropenem for group III strains. The MICs of beta-lactams for H. influenzae transformants into which the ftsI gene from BLNAR strains was introduced were as high as those for the donors, and PBP 3A and PBP 3B showed decreased affinities for beta-lactams. There was no clear relationship between 7-bp deletions in the dacB gene and AMP susceptibility. Even though mutations in another gene(s) may be involved in beta-lactam resistance, these data indicate that mutations in the ftsI gene are the most important for development of resistance to beta-lactams in BLNAR strains.
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PMID:Association of amino acid substitutions in penicillin-binding protein 3 with beta-lactam resistance in beta-lactamase-negative ampicillin-resistant Haemophilus influenzae. 1135 13

The crystal structure of YecO from Haemophilus influenzae (HI0319), a protein annotated in the sequence databases as hypothetical, and that has not been assigned a function, has been determined at 2.2-A resolution. The structure reveals a fold typical of S-adenosyl-L-methionine-dependent (AdoMet) methyltransferase enzymes. Moreover, a processed cofactor, S-adenosyl-L-homocysteine (AdoHcy), is bound to the enzyme, further confirming the biochemical function of HI0319 and its sequence family members. An active site arginine, shielded from bulk solvent, interacts with an anion, possibly a chloride ion, which in turn interacts with the sulfur atom of AdoHcy. The AdoHcy and nearby protein residues delineate a small solvent-excluded substrate binding cavity of 162 A(3) in volume. The environment surrounding the cavity indicates that the substrate molecule contains a hydrophobic moiety and an anionic group. Many of the residues that define the cavity are invariant in the HI0319 sequence family but are not conserved in other methyltransferases. Therefore, the substrate specificity of YecO enzymes is unique and differs from the substrate specificity of all other methyltransferases sequenced to date. Examination of the Enzyme Commission list of methyltransferases prompted a manual inspection of 10 possible substrates using computer graphics and suggested that the ortho-substituted benzoic acids fit best in the active site.
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PMID:Crystal structure of YecO from Haemophilus influenzae (HI0319) reveals a methyltransferase fold and a bound S-adenosylhomocysteine. 1174 87

Bacteriophage phiKZ is a giant virus that efficiently infects Pseudomonas aeruginosa strains pathogenic to human and, therefore, it is attractive for phage therapy. We present here the complete phiKZ genome sequence and a preliminary analysis of its genome structure. The 280,334 bp genome is a linear, circularly permutated and terminally redundant, A+T-rich double-stranded DNA molecule. The phiKZ DNA has no detectable sequence homology to other viruses and microorganisms, and it does not contain NotI, PstI, SacI, SmaI, XhoI, and XmaIII endonuclease restriction sites. The genome has 306 open reading frames (ORFs) varying in size from 50 to 2237 amino acid residues. According to the orientation of transcription, ORFs are apparently organized into clusters and most have a clockwise direction. The phiKZ genome also encodes six tRNAs specific for Met (AUG), Asn (AAC), Asp (GAC), Leu (TTA), Thr (ACA), and Pro (CCA). A putative promoter sequence containing a TATATTAC block was identified. Most potential stem-loop transcription terminators contain the tetranucleotide UUCG loops. Some genes may be assigned as phage-encoded RNA polymerase subunits. Only 59 phiKZ gene products exhibit similarity to proteins of known function from a diversity of organisms. Most of these conserved gene products, such as dihydrofolate reductase, ribonucleoside diphosphate reductase, thymidylate synthase, thymidylate kinase, and deoxycytidine triphosphate deaminase are involved in nucleotide metabolism. However, no virus-encoded DNA polymerase, DNA replication-associated proteins, or single-stranded DNA-binding protein were found based on amino acid homology, and they may therefore be strongly divergent from known homologous proteins. Fifteen phiKZ gene products show homology to proteins of pathogenic organisms, including Mycobacterium tuberculosis, Haemophilus influenzae, Listeria sp., Rickettsia prowazakeri, and Vibrio cholerae that must be considered before using this phage as a therapeutic agent. The phiKZ coat contains at least 40 polypeptides, and several proteins are cleaved during virus assembly in a way similar to phage T4. Eleven phiKZ-encoded polypeptides are related to proteins of other bacteriphages that infect a variety of hosts. Among these are four gene products that contain a putative intron-encoded endonuclease harboring the H-N-H motif common to many double-stranded DNA phages. These observations provide evidence that phages infecting diverse hosts have had access to a common genetic pool. However, limited homology on the DNA and protein levels indicates that bacteriophage phiKZ represents an evolutionary distinctive branch of the Myoviridae family.
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PMID:The genome of bacteriophage phiKZ of Pseudomonas aeruginosa. 1191 76

l-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) lies at the first branch point in the aspartate metabolic pathway that leads to the formation of the amino acids lysine, isoleucine, methionine, and threonine in most plants, bacteria, and fungi. Since the aspartate pathway is not found in humans, but is necessary for bacterial cell wall biosynthesis, the enzymes in this pathway are potential targets for the development of new antibiotics. The asd gene that encodes for ASA DH has been obtained from several infectious organisms and ligated into a pET expression vector. ASA DHs from Haemophilus influenza, Pseudomonas aeruginosa, and Vibrio cholerae were expressed as soluble proteins in Escherichia coli, while ASA DH from Helicobacter pylori was obtained primarily as inclusion bodies. The V. cholerae genome contains two asd genes. Both enzymes have been expressed and purified, and each displays significant ASA DH activity. The purification of highly active ASA DH from each of these organisms has been achieved for the first time, in greater than 95% purity and high overall yield. Kinetic parameters have been determined for each purified enzyme, and the values have been compared to those of E. coli ASA DH.
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PMID:Expression and purification of aspartate beta-semialdehyde dehydrogenase from infectious microorganisms. 1207 15

The enzyme tRNA(m(1)G37)methyltransferase (TrmD) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) specifically to guanosine at position 37 within a subset of tRNA species in bacteria. The modified guanosine is next to the anticodon and is important for the maintenance of the correct reading frame during translation. TrmD from Haemophilus influenzae with both N- and C-terminal tags was overexpressed in Escherichia coli and crystallized at 297 K using sodium acetate as a precipitant. Native X-ray diffraction data were collected to 1.85 A resolution. The crystals are rhombohedral, belonging to the space group R32, with unit-cell parameters a = b = 98.05, c = 176.79 A, alpha = beta = 90, gamma = 120 degrees. The presence of one monomer of recombinant TrmD in the crystallographic asymmetric unit gives a V(M) of 3.07 A(3) Da(-1) and a solvent content of 59.9%.
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PMID:Crystallization and preliminary X-ray crystallographic analysis of tRNA(m1G37)methyltransferase from Haemophilus influenzae. 1249 65

tRNA(m(1)G37)methyltransferase (TrmD) catalyzes the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to G(37) within a subset of bacterial tRNA species, which have a G residue at the 36th position. The modified guanosine is adjacent to and 3' of the anticodon and is essential for the maintenance of the correct reading frame during translation. Here we report four crystal structures of TrmD from Haemophilus influenzae, as binary complexes with either AdoMet or S-adenosyl-L-homocysteine (AdoHcy), as a ternary complex with AdoHcy and phosphate, and as an apo form. This first structure of TrmD indicates that it functions as a dimer. It also suggests the binding mode of G(36)G(37) in the active site of TrmD and the catalytic mechanism. The N-terminal domain has a trefoil knot, in which AdoMet or AdoHcy is bound in a novel, bent conformation. The C-terminal domain shows structural similarity to trp repressor. We propose a plausible model for the TrmD(2)-tRNA(2) complex, which provides insights into recognition of the general tRNA structure by TrmD.
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PMID:Crystal structure of tRNA(m1G37)methyltransferase: insights into tRNA recognition. 1277 76

The reversible dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde (ASA) in the aspartate biosynthetic pathway is catalyzed by aspartate-beta-semialdehyde dehydrogenase (ASADH). The product of this reaction is a key intermediate in the biosynthesis of diaminopimelic acid, an integral component of bacterial cell walls and a metabolic precursor of lysine and also a precursor in the biosynthesis of threonine, isoleucine and methionine. The structures of selected Haemophilus influenzae ASADH mutants were determined in order to evaluate the residues that are proposed to interact with the substrates ASA or phosphate. The substrate Km values are not altered by replacement of either an active-site arginine (Arg270) with a lysine or a putative phosphate-binding group (Lys246) with an arginine. However, the interaction of phosphate with the enzyme is adversely affected by replacement of Arg103 with lysine and is significantly altered when a neutral leucine is substituted at this position. A conservative Glu243 to aspartate mutant does not alter either ASA or phosphate binding, but instead results in an eightfold increase in the Km for the coenzyme NADP. Each of the mutations is shown to cause specific subtle active-site structural alterations and each of these changes results in decreases in catalytic efficiency ranging from significant (approximately 3% native activity) to substantial (<0.1% native activity).
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PMID:The role of substrate-binding groups in the mechanism of aspartate-beta-semialdehyde dehydrogenase. 1527 61


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