Cloning and nucleotide sequence of a bacterial cytochrome P-450VD25 gene encoding vitamin D-3 25-hydroxylase.
The gene encoding an enzyme that catalyzes the hydroxylation at position 25 of vitamin D-3 was cloned from an actinomycete strain, Amycolata autotrophica, by use of a host-vector system of Streptomyces lividans. The amino acid sequence deduced from the nucleotide sequence revealed that this enzyme, tentatively named P-450VD25, contains several regions of strong similarity with amino acid sequences of cytochromes P-450 from a variety of organisms, primarily in the regions of an oxygen-binding site and a heme ligand pocket. Especially, P-450VD25 shows end-to-end similarity in amino acid sequence to P-450dNIR of Fusarium oxysporum and P-450SU2 of Streptomyces griseolus. The recombinant S. lividans strain containing the P-450VD25 gene on a multicopy plasmid converted vitamin D-3 in the medium into 25-hydroxyvitamin D-3 at a maximum yield of 10%.
Kawauchi
H
H
Research Center, Taisho Pharmaceutical Co., Ltd., Saitama, Japan.
Sasaki
J
J
Adachi
T
T
Hanada
K
K
Beppu
T
T
Horinouchi
S
S
D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002112:Calcifediol / Q000096:biosynthesis; D002762:Cholecalciferol / Q000378:metabolism; D053493:Cholestanetriol 26-Monooxygenase; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013250:Steroid Hydroxylases / Q000235:genetics*
D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase; D002112:Calcifediol