Fatty acid biosynthesis in αand γ-proteobacteria requires two functionally distinct dehydratases, FabA and FabZ. Here, mechanistic cross-linking facilitates the structural characterization of a stable hexameric complex of six Escherichia coli FabZ dehydratase subunits with six AcpP acyl carrier proteins. The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is preorganized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization. de novo unsaturated fatty acid biosynthesis | cell membrane homeostasis | chemical biology | structural biology | computational biology Author contributions: J.A.M., J.L.S., and M.D.B. designed research; G.J.D., A.P., and K.L.J. performed research; J.A.M., J.L.S., and M.D.B. contributed new reagents/analytic tools;

A sophisticated intracellular trafficking pathway in humans is used to tailor vitamin B12 into its active cofactor forms, and to deliver it to two known B12-dependent enzymes. Herein, we report an unexpected strategy for cellular retention of B12, an essential and reactive cofactor. If methylmalonyl-CoA mutase is unavailable to accept the coenzyme B12 product of adenosyltransferase, the latter catalyzes homolytic scission of the cobalt−carbon bond in an unconventional reversal of the nucleophilic displacement reaction that was used to make it. The resulting homolysis product binds more tightly to adenosyltransferase than does coenzyme B12, facilitating cofactor retention. We have trapped, and characterized spectroscopically, an intermediate in which the cobalt−carbon bond is weakened prior to being broken. The physiological relevance of this sacrificial catalytic activity for cofactor retention is supported by the significantly lower coenzyme B12 concentration in patients with dysfunctional methylmalonyl-CoA mutase but normal adenosyltransferase activity.

Polyketide synthase (PKS) enzymes continue to hold great promise as synthetic biology platforms for the production of novel therapeutic agents, biofuels and commodity chemicals. Dehydratase (DH) catalytic domains play an important role during polyketide biosynthesis through the dehydration of the nascent polyketide intermediate to provide olefins. Our understanding of the detailed mechanistic and structural underpinning of DH domains that control substrate specificity and selectivity remains limited, thus hindering our efforts to rationally re-engineer PKSs. The curacin pathway houses a rare plurality of possible double bond permutations containing conjugated olefins as well as both cis- and trans-olefins, providing an unrivaled model system for polyketide dehydration. All four DH domains implicated in curacin biosynthesis were characterized in vitro using synthetic substrates and activity was measured by LC-MS/MS analysis. These studies resulted in complete kinetic characterization of the all trans trienoate-forming CurK dehydratase, whose kcat of 72 s−1 is more than three-orders of magnitude greater than any previously reported PKS DH domain. A novel stereospecific mechanism for diene formation involving a vinylogous enolate intermediate is proposed for the CurJ and CurH dehydratases based on incubation studies with truncated substrates. A synthetic substrate was co-crystallized with a catalytically inactive Phe substitution in the His-Asp catalytic dyad of CurJ DH to elucidate substrate-enzyme interactions. The resulting complex suggested the structural basis for dienoate formation and provided the first glimpse into the enzyme-substrate interactions essential for the formation of olefins in polyketide natural products. This examination of both canonical and non-canonical dehydration mechanisms reveals hidden catalytic activity inherent in some DH domains that may be leveraged for future applications in synthetic biology.

Metabolic engineering of polyketide synthase (PKS) pathways represents a promising approach to natural products discovery. The dehydratase (DH) domains of PKSs, which generate an α,β-unsaturated bond through a dehydration reaction, have been poorly studied compared to other domains, likely due to the simple nature of the chemical reaction they catalyze and lack of a convenient assay to measure substrate turnover. Herein we report the first steady-state kinetic analysis of a PKS DH domain employing LC-MS/MS analysis for product quantitation. PikDH2 was selected as a model DH domain. Its substrate specificity and mechanism was interrogated with a systematic series of synthetic triketide substrates containing a nonhydrolyzable thioether linkage as well as by site-directed mutagenesis, evaluation of the pH dependence of catalytic efficiency (Vmax/KM), and through kinetic characterization of a mechanism-based inhibitor. These studies revealed PikDH2 converts d-alcohol substrates to trans-olefin products. The reaction was reversible with equilibrium constants ranging from 1.2–2. Moreover, the enzyme activity was robust and PikDH2 was used on a preparative scale for the chemoenzymatic synthesis of unsaturated triketide products. PikDH2 was shown to possess remarkably strict substrate specificity and was unable to turnover substrates epimeric at the β, γ or δ-positions. We also demonstrated PikDH2 has a key ionizable group with a pKa of 7.0 and can be irreversibly inactivated through covalent modification by a mechanism-based inhibitor, which provides a foundation for future structural studies to elucidate substrate–protein interactions.

Covering: up to the end of 2018 Polyketides are a valuable source of bioactive and clinically important molecules. The biosynthesis of these chemically complex molecules has led to the discovery of equally complex polyketide synthase (PKS) pathways. Crystallography has yielded snapshots of individual catalytic domains, di-domains, and multi-domains from a variety of PKS megasynthases, and cryo-EM studies have provided initial views of a PKS module in a series of defined biochemical states. Here, we review the structural and biochemical results that shed light on the protein-protein interactions critical to catalysis by PKS systems with an embedded acyltransferase. Interactions include those that occur both within and between PKS modules, as well as with accessory enzymes.