Purification and characterization of the Mycobacterium tuberculosis FabD2, a novel malonyl-CoA:AcpM transacylase of fatty acid synthase.
Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in fatty acid and mycolic acid biosynthesis of Mycobacterium tuberculosis. fabd2 is a novel gene coding MCAT in M. tuberculosis besides another known fabd. In our study, fabd2 was inserted into a bacterial expression vector pET28a resulting in a 6x Histidine-tag fabd2 fusion gene construction. The protein was purified by nickel affinity chromatography and the characterizations of FabD2 have been investigated. The molecular weight of FabD2 was estimated to be 26 kDa by MALDI-TOF. Consistent with the biosynthesis specialty of reported MCATs, FabD2 resulted in a typical activity of bacterial MCATs, which catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP. Some physical and chemical differences between FabD2 and FabD also have been found. FabD2 shows dissimilarity with FabD in secondary structure in different pH buffer and MCAT genes RT-PCR results reveal different transcript condition with each other. Furthermore, FabD2 shows low similarity in protein sequence when alignment with other MCATs.
Huang
Yi-Shu
YS
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, PR China.
Ge
Jing
J
Zhang
Hong-Mei
HM
Lei
Jian-Qiang
JQ
Zhang
Xue-Lian
XL
Wang
Hong-Hai
HH
D051080:Acyl-Carrier Protein S-Malonyltransferase / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002413:Cations, Divalent / Q000737:chemistry; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D002413:Cations, Divalent; D011993:Recombinant Fusion Proteins; D051080:Acyl-Carrier Protein S-Malonyltransferase; D064429:Fatty Acid Synthases