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

ABSTRACT: Alkyl branching at the ? 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 ?-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 (Kd = 0.5 ?M) and could not be substituted by the acceptor ACP. High-resolution 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.

SUBMITTER: Maloney FP

PROVIDER: S-EPMC5027445 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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

Maloney Finn P FP Gerwick Lena L Gerwick William H WH Sherman David H DH Smith Janet L JL

Proceedings of the National Academy of Sciences of the United States of America 20160829 37

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 estab ...[more]

PMID: 27573844

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Project description:BackgroundACYL-LIPID THIOESTERASES (ALTs) are a subclass of plastid-localized, fatty acyl-acyl carrier protein (ACP) thioesterase enzymes from plants. They belong to the single hot dog-fold protein family. ALT enzymes generate medium-chain (C6-C14) and C16 fatty acids, methylketone precursors (β-keto fatty acids), and 3-hydroxy fatty acids when expressed heterologously in E. coli. The diverse substrate chain-length and oxidation state preferences of ALTs set them apart from other plant acyl-ACP thioesterases, and ALTs show promise as metabolic engineering tools to produce high-value medium-chain fatty acids and methylketones in bacterial or plant systems. Here, we used a targeted motif-swapping approach to explore connections between ALT protein sequence and substrate specificity. Guided by comparative motif searches and computational modelling, we exchanged regions of amino acid sequence between ALT-type thioesterases from Arabidopsis thaliana, Medicago truncatula, and Zea mays to create chimeric ALT proteins.ResultsComparing the activity profiles of chimeric ALTs in E. coli to their wild-type counterparts led to the identification of interacting regions within the thioesterase domain that shape substrate specificity and enzyme activity. Notably, the presence of a 31-CQH[G/C]RH-36 motif on the central α-helix was shown to shift chain-length specificity towards 12-14 carbon chains, and to be a core determinant of substrate specificity in ALT-type thioesterases with preference for 12-14 carbon 3-hydroxyacyl- and β-ketoacyl-ACP substrates. For an ALT containing this motif to be functional, an additional 108-KXXA-111 motif and compatible sequence spanning aa77-93 of the surrounding β-sheet must also be present, demonstrating that interactions between residues in these regions of the catalytic domain are critical to thioesterase activity. The behaviour of chimeric enzymes in E. coli also indicated that aa77-93 play a significant role in dictating whether an ALT will prefer ≤10-carbon or ≥ 12-carbon acyl chain-lengths, and aa91-96 influence selectivity for substrates of fully or partially reduced oxidation states. Additionally, aa64-67 on the hot dog-fold β-sheet were shown to be important for enabling an ALT to act on 3-hydroxy fatty acyl-ACP substrates.ConclusionsBy revealing connections between thioesterase sequence and substrate specificity, this study is an advancement towards engineering recombinant ALTs with product profiles suited for specific applications.

Dataset Information

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

Publications

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

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