Mycolic acid biosynthesis and enzymic characterization of the beta-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis.
Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a beta-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA.
Kremer
Laurent
L
Department of Microbiology and Immunology, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK.
Dover
Lynn G
LG
Carrère
Séverine
S
Nampoothiri
K Madhavan
KM
Lesjean
Sarah
S
Brown
Alistair K
AK
Brennan
Patrick J
PJ
Minnikin
David E
DE
Locht
Camille
C
Besra
Gurdyal S
GS
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D054889:Fatty Acid Synthase, Type II; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009171:Mycolic Acids / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D009097:Multienzyme Complexes; D009171:Mycolic Acids; D000123:Acetyltransferases; D054889:Fatty Acid Synthase, Type II