Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate

Abstract

Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (K d = 0.5 μM) and could not be substituted by the acceptor ACP. Highresolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre-and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA.natural products | polyketide synthase | curacin | HMG synthase | acyl carrier protein P olyketides are a large and chemically diverse group of natural products that includes many pharmaceuticals with a broad range of biological activities and applications as antibiotics, antifungals, antiinflammatory drugs, and cancer chemotherapeutic agents (1). Polyketide synthase (PKS) biosynthetic pathways are subjects of efforts to engineer diversification of natural products in pursuit of pharmaceutical leads and compounds of industrial importance (2). They are rich sources for development of chemoenzymatic catalysts based on PKS enzymes with unusual catalytic activities.Modular type I PKS pathways, among the most versatile of nature's systems, are biosynthetic assembly lines composed of modules that act in a defined sequence to produce complex products with a variety of functional groups and chiral centers. Each module is a set of fused catalytic domains that extend and modify a polyketide intermediate. Biosynthesis proceeds from intermediates tethered to acyl carrier protein (ACP) domains via a thioester link to a phosphopantetheine (Ppant) cofactor. A ketosynthase (KS) domain catalyzes extension of the intermediate, and subsequent modification domains typically catalyze reduction and/or methylation of the β-keto (3-keto) extension product. Beyond the enzymes for these core reactions, many PKS pathways also include other catalytic functionality. Among the most interesting of these noncanonical capabilities is alkylation at the β position by a set of β...

Chemoenzymatic Dissection of Polyketide β-Branching in the Bryostatin Pathway

“…Upon binding of ACP A , HMGS catalyzes an aldol addition of the α carbon of the acetyl unit to the β ketone carbon, resulting in an HMG-like product. Unlike HMG-CoA synthase that acts on CoA-linked substrates, the activity of HMGS has been found to be very specific to ACP A and ACP D , with little tolerance for substitution (Buchholz et al, 2010; Maloney, Gerwick, Gerwick, Sherman, & Smith, 2016). This ACP selectivity presents a challenge when the correct ACP is unknown, but may be overcome by use of surrogate carrier proteins that are closely related phylogenetically (Buchholz et al, 2010).…”

“…3), including mAcpC from the mupirocin pathway (El-Sayed et al, 2003) (40.5% identity) and CurB from the curacin pathway (43.0% identity), both of which were used in previous studies of BryR function. Comparison of BryU and BryR to a cocrystal structure of CurB interacting with the curacin HMGS (CurD, 67.5% identity to BryR) shows that the key residues for HMGS–ACP D interaction are conserved in both pathways (Maloney et al, 2016). This is perhaps to be expected given the relatively high sequence identity and binding affinity of CurB for BryR as a surrogate ACP D (Buchholz et al, 2010).…”

“…HMGS has been shown in several pathways, including bryostatin, to have a high degree of selectivity for both donor and acceptor ACPs. The basis of this selectivity has been described structurally in the curacin pathway, and the residues that are key to this binding mode appear to be conserved in both BryR (D214, D222, E225, and R266, CurD numbering) and BryU (R42, I46, and M47, CurB numbering) (Maloney et al, 2016). While the affinity of the curacin donor ACP is far tighter for its native HMGS than for BryR, the measured affinity still suggests that many of these donor ACPs have high degrees of intersystem operability (Buchholz et al, 2010).…”

“…BryT, CurE, and JamI (ECH 1 ) were expressed and purified using methods described for CurD (Maloney et al, 2016)…”

“…Apo-ACP was loaded with HMG-phosphopantetheine using HMG-CoA, Svp, and MgCl 2 using methods previously described (Maloney et al, 2016)…”