Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: Purification of acyl carrier protein (ACP) and malonyl-coenzyme A:ACP transacylase (FabD)

Alecksandr J. Kutchma, Tung T. Hoang, Herbert P. Schweizer

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

Abstract

A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding β-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding β-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled. The fabH gene (encoding β-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD, is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption-ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4'- phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H6-FabD exhibited malonyl-CoA:ACP transacylase activity.

Original languageEnglish (US)
Pages (from-to)5498-5504
Number of pages7
JournalJournal of Bacteriology
Volume181
Issue number17
DOIs
StatePublished - Sep 1999
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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