Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase.
Lipoic acid is a sulfur-containing 8-carbon fatty acid that functions as a central cofactor in multienzyme complexes that are involved in the oxidative decarboxylation of glycine and several alpha-keto acids. In its functional form, it is bound covalently in an amide linkage to the epsilon-amino group of a conserved lysine residue of the "lipoyl bearing subunit," resulting in a long "swinging arm" that shuttles intermediates among the requisite active sites. In Escherichia coli and many other organisms, the lipoyl cofactor can be synthesized endogenously. The 8-carbon fatty-acyl chain is constructed via the type II fatty acid biosynthetic pathway as an appendage to the acyl carrier protein (ACP). Lipoyl(octanoyl)transferase (LipB) transfers the octanoyl chain from ACP to the target lysine acceptor, generating the substrate for lipoyl synthase (LS), which subsequently catalyzes insertion of both sulfur atoms into the C-6 and C-8 positions of the octanoyl chain. In this study, we present a three-step isolation procedure that results in a 14-fold purification of LipB to >95% homogeneity in an overall yield of 25%. We also show that the protein catalyzes the transfer of the octanoyl group from octanoyl-ACP to apo-H protein, which is the lipoyl bearing subunit of the glycine cleavage system. The specific activity of the purified protein is 0.541 U mg(-1), indicating a turnover number of approximately 0.2 s(-1), and the apparent Km values for octanoyl-ACP and apo-H protein are 10.2+/-4.4 and 13.2+/-2.9 microM, respectively.
Nesbitt
Natasha M
NM
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Baleanu-Gogonea
Camelia
C
Cicchillo
Robert M
RM
Goodson
Kathy
K
Iwig
David F
DF
Broadwater
John A
JA
Haas
Jeffrey A
JA
Fox
Brian G
BG
Booker
Squire J
SJ
D000213:Acyl Carrier Protein / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000594:Amino Acid Oxidoreductases; D001059:Apoproteins / Q000378:metabolism; D002352:Carrier Proteins; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008954:Models, Biological; D008970:Molecular Weight; D009097:Multienzyme Complexes; D009994:Osmolar Concentration; D010957:Plasmids; D016133:Polymerase Chain Reaction; D020836:Protein Structure, Quaternary; D021241:Spectrometry, Mass, Electrospray Ionization; D014166:Transferases
D000213:Acyl Carrier Protein; D001059:Apoproteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C064525:glycine cleavage system; D006639:Histidine; D000594:Amino Acid Oxidoreductases; D014166:Transferases; D000217:Acyltransferases; C421483:LipB protein, E coli