Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: purification of acyl carrier protein (ACP) and malonyl-coenzyme A:ACP transacylase (FabD).
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 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 beta-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 H(6)-FabD exhibited malonyl-CoA:ACP transacylase activity.
Kutchma
A J
AJ
Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.
Hoang
T T
TT
Schweizer
H P
HP
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics / Q000378:metabolism; D000213:Acyl Carrier Protein / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D005227:Fatty Acids / Q000096:biosynthesis*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics
C101898:AcpP protein, Streptomyces; D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005227:Fatty Acids; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000217:Acyltransferases; D051080:Acyl-Carrier Protein S-Malonyltransferase; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase