Structural and Functional Studies of the Daunorubicin Priming Ketosynthase DpsC

Abstract

Daunorubicin is a type II polyketide, one of a large class of polyaromatic natural products with anticancer, antibiotic, and antiviral activity. Type II polyketides are formed by the assembly of malonyl-CoA building blocks, though in rare cases, biosynthesis is initiated by the incorporation of a nonmalonyl derived starter unit, which adds molecular diversity to the poly-β-ketone backbone. Priming mechanisms for the transfer of novel starter units onto polyketide synthases (PKS) are still poorly understood. Daunorubicin biosynthesis incorporates a unique propionyl starter unit thought to be selected for by a subclass ("DpsC type") of priming ketosynthases (KS III). To date, however, no structural information exists for this subclass of KS III enzymes. Although selectivity for self-acylation with propionyl-CoA has previously been implied, we demonstrate that DpsC shows no discrimination for self-acylation or acyl-transfer to the cognate acyl carrier protein, DpsG with short acyl-CoAs. We present five crystal structures of DpsC, including apo-DpsC, acetyl-DpsC, propionyl-DpsC, butyryl-DpsC, and a cocrystal of DpsC with a nonhydrolyzable phosphopantetheine (PPant) analogue. The DpsC crystal structures reveal the architecture of the active site, the molecular determinants for catalytic activity and homology to O-malonyl transferases, but also indicate distinct differences. These results provide a structural basis for rational engineering of starter unit selection in type II polyketide synthases.

Polyketide β-branching: diversity, mechanism and selectivity

“…Methyl-3-pentenoyl-ACP FogA8 essentially comprises an unusual acyl starter unit for the remainder of the biosynthetic pathway, requiring a dedicated priming KASIII-like KS, analogous to DpsC, prior to 'classical' aromatic polyketide chain extension catalysed by the minimal PKS, FogA9 (CLF), FogA10 (KS) and FogA11 (ACP). 90 Interestingly, FogA11 (ACP) may be shared with a second KS/ CLF (FogN/FogO) in the same biosynthetic cluster that synthesises both of the major metabolites fogacin and fogacin B in a parallel pathway, both of which lack the trisubstituted exoalkene and share common biosynthetic intermediates with the classic aromatic polyketide actinorhodin. (Fig.…”

Involvement of β‐Alkylation Machinery and Two Sets of Ketosynthase‐Chain‐Length Factors in the Biosynthesis of Fogacin Polyketides in Actinoplanes missouriensis

“…These FabH‐like enzymes were predicted to be important for the selection of the specific acyl starter substrate. DpsC was reported to catalyze either the first condensation between the starter and extender substrates or the transfer of a specific starter acyl moiety to the ACP of a minimal PKS, or both, but it still remains unclear whether DpsC catalyzes either or both of these two reactions under physiological conditions . Anyway, we speculate that FogA12 has an important role in the selection of the specific acyl starter substrate by minimal PKS FogA9‐11.…”

“…We speculate that FogA11 (ACP), FogA10 (KS), and FogA9 (CLF) compose a minimal PKS and that FogA12 (KAS III), FogA8 (ACP), and FogA2 (KS) have some important roles in HCS cassettedependent β‐alkylated polyketide synthesis by the minimal type II PKS. FogA12 (KAS III) is similar to AknE2 (47 %) and DpsC (45 %), which are FabH‐like enzymes encoded in type II PKS gene clusters. These FabH‐like enzymes were predicted to be important for the selection of the specific acyl starter substrate.…”

Discovery and biosynthesis of gladiochelins: unusual lipodepsipeptide siderophores fromBurkholderia gladioli

“…The functional significance of this is unclear, but an analogous Cys to Ser mutation is observed in DpsC, a KAS III homologue that has been reported to initiate assembly of the daunorubicin polyketide chain using a propionyl-CoA starter unit. 46 Further processing of the -ketothioester resulting from elongation of (O-methyl)-citryl-CoA with malonyl-ACP by the primary metabolic FAS would afford the saturated O-methyl-citrate-primed fatty acyl-ACP thioester 10 (Figure 3). The 11, 12 and 2, 3 double bonds are likely introduced into this thioester by GcnF and GcnL, which are similar to membrane-associated fatty acid desaturases and acyl-ACP desaturases, respectively ( Figure 3 and Table 1), completing the assembly of the unusual fatty acid residue incorporated into the gladiochelins.…”