Enzymatic production of (-)-indolactam V by LtxB, a cytochrome P450 monooxygenase.
The P450 cytochrome monooxygenase gene, ltxB, was cloned and overexpressed in Escherichia coli as a 6xHis-tagged protein. The resulting recombinant LtxB was purified by Ni-NTA affinity chromatography and characterized biochemically. Purified LtxB demonstrated typical cytochrome P450 spectroscopic properties including substrate-induced transition from a low-spin (lambdamax=414 nm) to high-spin state (lambdamax=386 nm) upon incubation with N-methyl-L-valyl-L-tryptophanol. The catalytic activity of LtxB was verified by demonstrating the oxidation/cyclization of N-methyl-L-valyl-L-tryptophanol to (-)-indolactam V. LtxB shows a relaxed specificity for analogue substrates in which the valyl group is substituted for other aliphatic groups. The relaxed substrate specificity of LtxB, along with the relaxed specificity of the prenyltransferase, LtxC, allowed for the enzymatic production of a series of (-)-indolactam V and lyngbyatoxin analogues.
Huynh
Minh U
MU
Department of Chemistry and Biochemistry, California State University, Chico, 400 W. 1st Street, Chico, California 95929, USA.
Elston
Matthew C
MC
Hernandez
Nick M
NM
Ball
David B
DB
Kajiyama
Shin-ichiro
S
Irie
Kazuhiro
K
Gerwick
William H
WH
Edwards
Daniel J
DJ
D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007211:Indoles / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007769:Lactams / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008235:Lyngbya Toxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007211:Indoles; D007769:Lactams; D008235:Lyngbya Toxins; D011994:Recombinant Proteins; C043969:indolactam V; D003577:Cytochrome P-450 Enzyme System