Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II.
Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA:AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.
Kremer
L
L
INSERM U447, Institut Pasteur de Lille, 1 rue du Pr. Calmette, BP245-59019 Lille Cedex, France.
Nampoothiri
K M
KM
Lesjean
S
S
Dover
L G
LG
Graham
S
S
Betts
J
J
Brennan
P J
PJ
Minnikin
D E
DE
Locht
C
C
Besra
G S
GS
D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D064429:Fatty Acid Synthases / Q000737:chemistry*; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010957:Plasmids / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship; D013696:Temperature
C431823:AcpM protein, Mycobacterium tuberculosis; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D000217:Acyltransferases; D051080:Acyl-Carrier Protein S-Malonyltransferase; C494968:fabD protein, E coli; D064429:Fatty Acid Synthases; D054889:Fatty Acid Synthase, Type II