Expression and characterization of the type III polyketide synthase 1,3,6,8-tetrahydroxynaphthalene synthase from Streptomyces coelicolor A3(2).
Sequence analysis of the metabolically rich 8.7-Mbp genome of the model actinomycete Streptomyces coelicolor A3(2) revealed three genes encoding predicted type III polyketide synthases (PKSs). We report the inactivation, expression, and characterization of the type III PKS homologous SCO1206 gene product as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Incubation of recombinant THNS with malonyl-CoA showed THN production, as demonstrated by UV and HPLC analyses. The K(m) value for malonyl-CoA and the k(cat) value for THN synthesis were determined spectrophotometrically to be 3.58+/-0.85 micro M and 0.48+/-0.03 min(-1), respectively. The C-terminal region of S. coelicolor THNS, which is longer than most other bacterial and plant type III PKSs, was shortened by 25 amino acid residues and the resulting mutant was shown to be slightly more active (K(m)=1.97+/-0.19 micro M, k(cat)=0.75+/-0.04 min(-1)) than the wild-type enzyme.
Izumikawa
Miho
M
College of Pharmacy, University of Arizona, 1703 E. Mabel, Tucson, AZ 85721-0207, USA.
Shipley
Paul R
PR
Hopke
Jörn N
JN
O'Hare
Thomas
T
Xiang
Longkuan
L
Noel
Joseph P
JP
Moore
Bradley S
BS
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D007218:Industrial Microbiology; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis; D009284:Naphthols / Q000378:metabolism*; D010802:Phylogeny; D010858:Pigmentation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
C121288:1,3,6,8-tetrahydroxynaphthalene; D009097:Multienzyme Complexes; D009284:Naphthols; D011994:Recombinant Proteins; D000217:Acyltransferases; C020141:flavanone synthetase