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Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB).

ABSTRACT: The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemophilus influenzae. Chromosomal fabA and fabB mutants were isolated; the mutants were auxotrophic for unsaturated fatty acids. A temperature-sensitive fabA mutant was obtained by site-directed mutagenesis of a single base that induced a G101D change; this mutant grew normally at 30 degrees C but not at 42 degrees C, unless the growth medium was supplemented with oleate. By physical and genetic mapping, the fabAB genes were localized between 3.45 and 3.6 Mbp on the 5.9-Mbp chromosome, which corresponds to the 58- to 59.5-min region of the genetic map.

SUBMITTER: Hoang TT

PROVIDER: S-EPMC179400 | biostudies-other | 1997 Sep

REPOSITORIES: biostudies-other

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Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB).

Hoang T T TT Schweizer H P HP

Journal of bacteriology 19970901 17

The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemophil ...[more]

PMID: 9286984

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Project description:Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.

Project description:Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential beta-ketoacyl-acyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr(191) being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr(191) alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

Dataset Information

Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB).

Publications

Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB).

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