In Vitro Reconstitution and Analysis of the Chain Initiating Enzymes of the R1128 Polyketide Synthase

Abstract

Biosynthesis of the carbon chain backbone of the R1128 substances is believed to involve the activity of a ketosynthase/chain length factor (ZhuB/ZhuA), an additional ketosynthase (ZhuH), an acyl transferase (ZhuC), and two acyl carrier proteins (ACPs; ZhuG and ZhuN). A subset of these proteins initiate chain synthesis via decarboxylative condensation between an acetyl-, propionyl-, isobutyryl-, or butyryl-CoA derived primer unit and a malonyl-CoA derived extender unit to yield an acetoacetyl-, beta-ketopentanoyl-, 3-oxo-4-methylpentanoyl-, or beta-ketohexanoyl-ACP product, respectively. To investigate the precise roles of ZhuH, ZhuC, ZhuG, and ZhuN, each protein was expressed in Escherichia coli and purified to homogeneity. Although earlier reports had proposed that ZhuC and its homologues played a role in primer unit selection, direct in vitro analysis of ZhuC showed that it was in fact a malonyl-CoA:ACP malonyltransferase (MAT). The enzyme could catalyze malonyl transfer but not acetyl- or propionyl-transfer onto R1128 ACPs or onto ACPs from other biosynthetic pathways, suggesting that ZhuC has broad substrate specificity with respect to the holo-ACP substrate but is specific for malonyl-CoA. Thus, ZhuC supplies extender units to both the initiating and elongating ketosynthases from this pathway. To interrogate the primer unit specificity of ZhuH, the kinetics of beta-ketoacyl-ACP formation in the presence of various acyl-CoAs and malonyl-ZhuG were measured. Propionyl-CoA and isobutyryl-CoA were the two most preferred substrates of ZhuH, although acetyl-CoA and butyryl-CoA could also be accepted and elongated. This specificity is not only consistent with earlier reports demonstrating that R1128B and R1128C are the major products of the R1128 pathway in vivo, but is also in good agreement with the properties of the ZhuH substrate binding pocket, as deduced from a recently solved crystal structure of the enzyme. Finally, to investigate the molecular logic for the occurrence of not one but two ACP genes within the R1128 gene cluster, the inhibition of ZhuH-catalyzed formation of beta-ketopentanoyl-ACP was quantified in the presence of apo-ZhuG or apo-ZhuN. Both apo-proteins were comparable inhibitors of the ZhuH catalyzed reaction, suggesting that the corresponding apo-proteins can be used interchangeably during chain initiation. Together, these results provide direct biochemical insights into the mechanism of chain initiation of an unusual bacterial aromatic PKS.

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

“…We previously showed that nonacetate-primed aromatic PKSs contain discrete initiation modules that typically consist of (i) a second ACP (ACP p ) that primes the minimal PKS with the nonacetate unit (34), (ii) a ketosynthase III that synthesizes the ACP p -bound primer unit (39), and (iii) a potent acetyl-ACP thiolase that hydrolyzes the competing acetyl-ACP species that may otherwise initiate polyketide assembly with acetate (56). An ORF (oxyP) 15 kb downstream of the minimal PKS genes encodes an enzyme of high sequence homology to acetyl-ACP thiolases, such as ZhuC from the R1128 PKS (34) (49% identity) and FrnK from the frenolicin PKS (1) (43% identity).…”

Engineered Biosynthesis of Aklanonic Acid Analogues

“…(Figure 3) 12,19,20,21 In some cases, such as the R1128 PKS and the daunorubicin PKS, the synthase includes a dedicated module of enzymes for chain initiation. 22,23 Recent studies have demonstrated that these initiation modules can collaborate with elongation modules from heterologous octaketide synthases and decaketide synthases to produce non-acetyl primed hexaketide and octaketide natural products, respectively. 24 In each case, chain length of the polyketide backbone is dictated by the elongation module (i.e., the minimal PKS).…”

Oxytetracycline Biosynthesis

“…As illustrated first by Khosla and co-workers (44), the oxy minimal PKS alone synthesizes exclusively acetateprimed decaketides. In other pathways in which nonacetate starter units are employed, such as daunorubicin (45), frenolicin (46), R1128 (46,47), and hedamycin (48,49), an additional initiation module has been implicated in starter unit selection. In the case of frenolicin and R1128, it was further shown that a dedicated ACP is required for the chain initiation steps and plays a different role than the ACP utilized by the minimal PKS for chain elongation (46).…”