Expression, purification and crystallization of the cofactor-independent monooxygenase SnoaB from the nogalamycin biosynthetic pathway.
12-deoxy-nogalonic acid oxygenase (SnoaB) catalyzes the oxygenation of 12-deoxy-nogalonic acid at position 12 to yield nogalonic acid, which is one of the steps in the biosynthesis of the polyketide nogalamycin in Streptomyces nogalater. SnoaB belongs to a family of small cofactor-free oxygenases which carry out oxygenation reactions without the aid of any prosthetic group, cofactor or metal ion. Recombinant SnoaB was crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 58.8, b = 114.1, c = 49.5 A, and these crystals diffracted to 2.4 A resolution. Recombinant SnoaB does not contain any methionine residues and three double mutants were designed and produced for the preparation of selenomethionine-substituted samples. The selenomethionine-substituted mutant F40M/L89M crystallized in the same space group as the native enzyme.
Koskiniemi
Hanna
H
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Grocholski
Thadee
T
Schneider
Gunter
G
Niemi
Jarmo
J
D000595:Amino Acid Sequence; D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009621:Nogalamycin / Q000096:biosynthesis* / Q000737:chemistry; D012645:Selenomethionine / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*
D003067:Coenzymes; D050505:Mutant Proteins; D012645:Selenomethionine; D006899:Mixed Function Oxygenases; D009621:Nogalamycin