Expression, purification, and characterization of the Mycobacterium tuberculosis acyl carrier protein, AcpM.
Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: the multifunctional polypeptide, FASI, in which the acyl carrier protein (ACP) domain forms an integral part of the polypeptide, and the dissociated FASII system, which is composed of monofunctional enzymes and a discrete ACP (AcpM). In order to characterize enzymes of the FASII system, large amounts of AcpM are required to generate substrates such as holo-AcpM, malonyl-AcpM and acyl-AcpM. The M. tuberculosis acpM gene was overexpressed in Escherichia coli and AcpM purified, yielding approximately 15-20 mg/l of culture. Analysis of AcpM by mass spectrometry, N-terminal sequencing, amino acid analysis, and gas chromatography indicated the presence of three species, apo-, holo-, and acyl-AcpM, the former comprising up to 65% of the total pool. The apo-AcpM was purified away from the in vivo generated holo- and acyl-forms, which were inseparable and heterogeneous with respect to acyl chain lengths. Once purified, we were able to convert apo-AcpM into holo- and acyl-forms. These procedures provide the means for the preparation of the large quantities of AcpM and derivatives needed for characterization of the purified enzymes of the mycobacterial FASII system.
Schaeffer
M L
ML
GlaxoSmithKline, Collegeville, PA 19426, USA. merrill_l_schaeffer@sbphrd.com
Agnihotri
G
G
Kallender
H
H
Brennan
P J
PJ
Lonsdale
J T
JT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D019730:Carbon-Sulfur Ligases / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002849:Chromatography, Gas; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
C431823:AcpM protein, Mycobacterium tuberculosis; D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D019730:Carbon-Sulfur Ligases; C018749:long-chain-fatty-acid-(acyl-carrier-protein) ligase