title: Enzymatic production of (-)-indolactam V by LtxB, a cytochrome P450 monooxygenase.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Chico, 400 W. 1st Street, Chico, California 95929, USA.
authors.forename: Minh U
authors.initials: MU
authors.lastname: Huynh
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Elston
authors.forename: Nick M
authors.initials: NM
authors.lastname: Hernandez
authors.forename: David B
authors.initials: DB
authors.lastname: Ball
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Kajiyama
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Irie
authors.forename: William H
authors.initials: WH
authors.lastname: Gerwick
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Edwards
mesh_terms: 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
chemical_list: D007211:Indoles; D007769:Lactams; D008235:Lyngbya Toxins; D011994:Recombinant Proteins; C043969:indolactam V; D003577:Cytochrome P-450 Enzyme System