Crystal structure of OxyB, a cytochrome P450 implicated in an oxidative phenol coupling reaction during vancomycin biosynthesis.
Gene-inactivation studies point to the involvement of OxyB in catalyzing the first oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. The oxyB gene has been cloned and sequenced from the vancomycin producer Amycolatopsis orientalis, and the hemoprotein has been produced in Escherichia coli, crystallized, and its structure determined to 1.7-A resolution. OxyB gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite or by spinach ferredoxin and ferredoxin reductase, the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. Addition of putative heptapeptide substrates to resting OxyB produced type I changes to the UV spectrum, but no turnover was observed in the presence of ferredoxin and ferredoxin reductase, showing that either the peptides or the reduction system, or both, are insufficient to support a full catalytic cycle. OxyB exhibits the typical P450-fold, with helix L containing the signature sequence FGHGXHXCLG and Cys(347) being the proximal axial thiolate ligand of the heme iron. The structural similarity of OxyB is highest to P450nor, P450terp, CYP119, and P450eryF. In OxyB, the F and G helices are rotated out of the active site compared with P450nor, resulting in a much more open active site, consistent with the larger size of the presumed heptapeptide substrate.
Institute of Organic Chemistry, University of Zurich, Switzerland.
Katja
K
Zerbe
Olena
O
Pylypenko
Francesca
F
Vitali
Weiwen
W
Zhang
Severine
S
Rouset
Markus
M
Heck
Jan W
JW
Vrijbloed
Daniel
D
Bischoff
Bojan
B
Bister
Roderich D
RD
Süssmuth
Stefan
S
Pelzer
Wolfgang
W
Wohlleben
John A
JA
Robinson
Ilme
I
Schlichting
D000192:Actinomycetales / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019800:Phenol / Q000378:metabolism; D011485:Protein Binding; D011987:Receptors, Steroid / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D013053:Spectrophotometry; D014466:Ultraviolet Rays; D014640:Vancomycin / Q000096:biosynthesis*; D014965:X-Rays; D000098825:Oxysterol Binding Proteins
D005288:Ferredoxins; D010455:Peptides; D011987:Receptors, Steroid; D000098825:Oxysterol Binding Proteins; D019800:Phenol; D014640:Vancomycin; D001216:Asparagine; D005287:Ferredoxin-NADP Reductase; D010100:Oxygen