Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: Purification of acyl carrier protein (ACP) and malonyl-coenzyme A : ACP transacylase (FabD)
Aj. Kutchma et al., Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: Purification of acyl carrier protein (ACP) and malonyl-coenzyme A : ACP transacylase (FabD), J BACT, 181(17), 1999, pp. 5498-5504
A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl
-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding
beta-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding beta-
ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene clu
ster is delimited by the pLsX (encoding a poorly understood enzyme of phosp
holipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) gen
es; the fabF and pabC genes seem to be translationally coupled. The fabH ge
ne (encoding beta-ketoacyl-ACP synthase III), which in most gram-negative b
acteria is located between plsX and fabD, is absent from this gene cluster.
A chromosomal temperature-sensitive fabD mutant was obtained by site-direc
ted mutagenesis that resulted in a W258Q change. A chromosomal fabF inserti
on mutant was generated, and the resulting mutant strain contained substant
ially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disru
ption 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 a
nd purification scheme that should be applicable to ACP from other bacteria
. Matrix-assisted laser desorption-ionization spectroscopy, native polyacry
lamide electrophoresis, and amino-terminal sequencing revealed that (i) mos
t of the purified ACP was properly modified with its 4'-phosphopantetheine
functional group, (ii) it was not acylated, and (iii) the amino-terminal me
thionine was removed. In an in vitro system, purified ACP functioned as acy
l acceptor and H-6-FabD exhibited malonyl-CoA:ACP transacylase activity.