CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime.
The cytochrome P450 CYP79B1 from Sinapis alba has been heterologously expressed in Escherichia coli and shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime. Three expression constructs were made, one expressing the native protein and two expressing proteins with different N-terminal modifications. The native construct gave the highest yield as estimated by enzymatic activity per liter of culture. Spheroplasts of E. coli expressing CYP79B1 were reconstituted with the Arabidopsis thaliana NADPH:cytochrome P450 reductase ATR1 heterologously expressed in E. coli to obtain enzymatic activity. This indicates that the E. coli electron-donating system, flavodoxin/flavodoxin reductase, does not support CYP79B1 activity. Recombinant CYP79B1 has a K(m) for tryptophan of 29+/-2 microM and a V(max) of 36.5+/-0.7nmolh(-1)(mlculture)(-1). The identity at the amino acid level of CYP79B1 is, respectively, 93 and 84% to CYP79B2 and CYP79B3 from A. thaliana, and 96% to CYP79B5 (Accession No. AF453287) from Brassica napus. The CYP79B subfamily of cytochromes P450 is likely to constitute a group of orthologous genes in the biosynthesis of indole glucosinolates.
Naur
Peter
P
Plant Biochemistry Laboratory, Department of Plant Biology and Center for Molecular Plant Physiology (PlaCe), The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg, Copenhagen, Denmark.
Hansen
Carsten Hørslev
CH
Bak
Søren
S
Hansen
Bjarne Gram
BG
Jensen
Niels Bjerg
NB
Nielsen
Hanne Linde
HL
Halkier
Barbara Ann
BA
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D001937:Brassica / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism / Q000502:physiology*; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D017830:Octoxynol; D010091:Oximes / Q000378:metabolism*; D010802:Phylogeny; D011092:Polyethylene Glycols / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D031226:Sinapis / Q000201:enzymology*; D013696:Temperature; D014364:Tryptophan / Q000378:metabolism*
D003902:Detergents; D007211:Indoles; D010091:Oximes; D011994:Recombinant Proteins; C467098:indole-3-acetaldoxime; D011092:Polyethylene Glycols; D014364:Tryptophan; D017830:Octoxynol; D003577:Cytochrome P-450 Enzyme System; C010615:Nonidet P-40; D006899:Mixed Function Oxygenases; C471052:cytochrome P-450 CYP79B1, Sinapis alba; C089817:cytochrome P450TYR