Stereochemical course of the hydration reaction catalysed by β-hydroxydecanoylthioester dehydrase

Abstract

Enzyme-catalysed hydration of the N-acetylcysteamine thioester of (E)-dec-2-enoic acid to the corresponding thioester of (R)-3-hydroxydecanoic acid proceeds in a syn fashion, with protonation on the si face at substrate C-2. P-Hydroxydecanoylthioester dehydrase , I the key enzyme in the biosynthesis of unsaturated fatty acids in bacteria under anaerobic conditions, mediates the interconversion of thioesters of (R)-3-hydroxydecanoic acid, (Q-dec-2-enoic acid, and (Z)-dec-3-enoic acid, (l), (2), and (3), respectively (R = acyl carrier protein, in vivo). We present evidence herein that the dehydrase-catalysed hydration of (2) to give (1) is a syn

Structural and dynamical rationale for fatty acid unsaturation in Escherichia coli

“…Additionally, E2 dehydrations require that the two groups being eliminated, here the C α hydrogen and β-hydroxy group, must be antiperiplanar to each other, characterized by a C (O) -C α -C β -C γ dihedral angle of 180°. Previous mechanistic studies of FabA and the crystal structure of P. aeruginosa FabA in complex with a substrate mimic support the E1cb-like mechanism (14)(15)(16)(17)(18). Recent mechanistic studies of a FabA homolog in a type I polyketide synthase was interpreted in terms of a "single-base" mechanism, in which the active His abstracts a proton from the substrate C α and also protonates the β-hydroxy leaving group.…”

“…Further, while the dehydration mechanism ( Fig. 1B) of FabA is known (15)(16)(17)(18), the mechanism of isomerization ( Fig. 1C), the second step in the transformation, remains speculative (12,17,19).…”

Polyketide stereocontrol: a study in chemical biology

“…The DHs catalyze the elimination of water from the polyketide intermediates to form double bonds which are typically trans ( E ) in configuration, although cis ( Z ) alkenes are also present in a significant fraction of structures [ 94 ]. Studies on the evolutionary related DHs from animal FAS which produce exclusively trans double bonds [ 96 ] have demonstrated that this reaction proceeds with overall syn elimination of the pro -(2 S ) hydrogen and the (3 R )-hydroxy group [ 97 – 98 ], while biochemical and stereochemical experiments on this class of enzymes suggest a catalytic mechanism in which a single histidine plays the role of both general acid and base [ 97 – 99 ]. Extending this proposal to PKS DHs which operate on C-2 methylated intermediates, implies that only D-methylated ((2 R ), unepimerized) compounds will be substrates for the DHs, as then the C-2 proton is of the correct stereochemistry.…”

“…In terms of the catalytic mechanism, a two-base mechanism has been proposed based on the crystal structure of the DEBS DH [ 92 ], in which the conserved His acts as a general base to deprotonate at C-2, while an Asp residue serves as a general acid to stabilize the C-3 hydroxy leaving group. However, only the His has been shown by site-directed mutagenesis to be essential [ 111 – 112 ], and so definitive proof of whether the classic one-base mechanism mentioned earlier [ 97 – 99 ] or alternative two-base mechanism applies [ 113 ], remains to be obtained.…”

Active Site Comparisons and Catalytic Mechanisms of the Hot Dog Superfamily

“…It also indicates that an acid/base mechanism is not in fact in play but that histidine acts as both base and acid (as histidinium) or that a concerted syn addition occurs, both of which agree with previous biochemical and stereochemical experiments on this class of enzymes. 8,9,48 …”