Catalytic relationships between type I and type II iterative polyketide synthases: The Aspergillus parasiticus norsolorinic acid synthase.
Norsolorinic acid synthase (NSAS) is a type I iterative polyketide synthase that occurs in the filamentous fungus Aspergillus parasiticus. PCR was used to clone fragments of NSAS corresponding to the acyl carrier protein (ACP), acyl transferase (AT) and beta-ketoacyl-ACP synthase (KS) catalytic domains. Expression of these gene fragments in Escherichia coli led to the production of soluble ACP and AT proteins. Coexpression of ACP with E. coli holo-ACP synthase (ACPS) let to production of NSAS holo-ACP, which could also be formed in vitro by using Streptomyces coelicolor ACPS. Analysis by mass spectrometry showed that, as with other type I carrier proteins, self-malonylation is not observed in the presence of malonyl CoA alone. However, the NSAS holo-ACP serves as substrate for S. coelicolor MCAT, S. coelicolor actinorhodin holo-ACP and NSAS AT domain-catalysed malonate transfer from malonyl CoA. The AT domain could transfer malonate from malonyl CoA to NSAS holo-ACP, but not hexanoate or acetate from either the cognate CoA or FAS ACP species to NSAS holo-ACP. The NSAS holo-ACP was also active in actinorhodin minimal PKS assays, but only in the presence of exogenous malonyl transferases.
Ma
Yue
Y
School of Chemistry, University of Bristol, Cantock's Close, Clifton, Bristol, BS8 1TS, UK.
Smith
Leah H
LH
Cox
Russell J
RJ
Beltran-Alvarez
Pedro
P
Arthur
Christopher J
CJ
Simpson F R S
Thomas J
TJ
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000737:chemistry; D001230:Aspergillus / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D008314:Malonates / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; D048630:Polyketide Synthases / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D000095622:Benzoisochromanequinones
D000429:Alcohol Oxidoreductases; D000880:Anthraquinones; D005656:Fungal Proteins; D008314:Malonates; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; D048630:Polyketide Synthases; C030290:malonic acid; C062609:norsolorinic acid ketoreductase; C013390:actinorhodin; D000095622:Benzoisochromanequinones