Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase.
BACKGROUND
Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC).


RESULTS
The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate.


CONCLUSIONS
Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.
Zawada
R J
RJ
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Khosla
C
C
D000213:Acyl Carrier Protein / Q000096:biosynthesis / Q000235:genetics; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D000446:Aldehyde-Lyases / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003500:Cyclization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D007202:Indicators and Reagents; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C087192:polyketide synthase ketoreductase; D000217:Acyltransferases; D051080:Acyl-Carrier Protein S-Malonyltransferase; C494968:fabD protein, E coli; D000446:Aldehyde-Lyases; C083875:tetracenomycin F2 cyclase, Streptomyces; D054889:Fatty Acid Synthase, Type II