Characterization of a Pseudomonas aeruginosa Fatty Acid Biosynthetic Gene Cluster: Purification of Acyl Carrier Protein (ACP) and Malonyl-Coenzyme A:ACP Transacylase (FabD)

• DOI: 10.1128/jb.181.17.5498-5504.1999
• Publication Date: 2019-12-31T16:38:37Z
• Authors: Alecksandr J. Kut...; Tung T. Hoang; Herbert P. Schweizer
• Keywords: DNA, Bacterial; 0301 basic medicine; 2. Zero hunger; Base Sequence; Fatty Acids; Molecular Sequence Data; Alcohol Oxidoreductases; 03 medical and health sciences; Bacterial Proteins; Genes, Bacterial; Multigene Family; 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Pseudomonas aeruginosa; Acyl Carrier Protein; Acyl-Carrier Protein S-Malonyltransferase; 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; Cloning, Molecular; Acyltransferases

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 (H 6 -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 H 6 -FabD exhibited malonyl-CoA:ACP transacylase activity.