Crotonases: Nature’s Exceedingly Convertible Catalysts

Abstract

The crotonases comprise a widely-distributed enzyme superfamily that has multiple roles in both primary and secondary metabolism. Many crotonases employ oxyanion holemediated stabilization of intermediates to catalyze the reaction of coenzyme A (CoA) thioester substrates (e.g., malonyl-CoA, α,β-unsaturated CoA esters) with both nucleophiles and, in the case of enolate intermediates, with varied electrophiles. Reactions of crotonases that proceed via a stabilized oxyanion intermediate include the hydrolysis of substrates including proteins, as well as hydration, isomerization, nucleophilic aromatic substitution, Claisen-type, and cofactor-independent oxidation reactions. The crotonases have a conserved fold formed from a central β-sheet core surrounded by α-helices, which typically oligomerizes to form a trimer, or dimer of trimers. The presence of a common structural platform and mechanisms involving intermediates with diverse reactivity implies that crotonases have considerable potential for biocatalysis and synthetic biology, as supported by pioneering protein engineering studies on them. In this Perspective article, we give an overview of crotonase diversity and structural biology, then illustrate the scope of crotonase catalysis and potential for biocatalysis.

Control of β‐Branching in Kalimantacin Biosynthesis: Application of 13C NMR to Polyketide Programming

“…Only the kalimantacin and phormidolide pathways are now known to have two ECH2 domains, and therefore either route is feasible. CS family members are notoriously difficult to functionally classify based on amino acid sequence or 3D architecture, and homology modelling did not suggest a likelihood of mECH catalysing either of these reactions.…”

“…The HCS cassette (BatA, C–E), modular ACP4‐6, modular ECH2 (mECH), and reductase (BatK) were recombinantly overexpressed in E. coli and purified (Figures S2 and S3). Both BatD and BatE were shown to be trimeric by size‐exclusion chromatography (SEC) . Pantetheine‐bound biosynthetic intermediates were prepared from 2 : Ac‐pantetheine 3, acetoacetyl (Acac)‐pantetheine 4 , HMG‐pantetheine 5 , 3‐methylbut‐2‐enoyl‐pantetheine 6 , and [2‐ 13 C]‐Ac‐pantetheine 8 (Scheme and Schemes S1–S3 and Material and Methods in the Supporting Information) and used as substrates for enzymatic reactions or converted chemoenzymatically into the corresponding CoAs for transfer onto apo‐ACP …”

“…This matches the point of exo-b-methylene incorporation and suggests that this modular ECH2 may control this step ( Figure 2B). [7] An exo-b-methylene may,h owever, arise through either the modular ECH2 intercepting the dehydrated product through decarboxylation and alternative reprotonation at the C-2 position before the trans-acting ECH2 can access it ( Figure 2B,i ), or the modular ECH2-catalyzed isomerisation of an endo-b-methyl branch ( Figure 2B,i i).O nly the kalimantacin and phormidolide pathways are now known to have two ECH2 domains,and therefore either route is feasible.CS family members are notoriously difficult to functionally classify based on amino acid sequence or 3D architecture, [8] and homology modelling did not suggest al ikelihood of mECH catalysing either of these reactions.…”

“…Both BatD and BatE were shown to be trimeric by sizeexclusion chromatography (SEC). [8] Pantetheine-bound biosynthetic intermediates were prepared from 2:A c-pantetheine 3, acetoacetyl (Acac)-pantetheine 4,H MG-pantetheine 5,3 -methylbut-2-enoyl-pantetheine 6,a nd [2-13 C]-Acpantetheine 8 (Scheme 1a nd Schemes S1-S3 and Material and Methods in the Supporting Information) and used as substrates for enzymatic reactions or converted chemoenzy-matically into the corresponding CoAs for transfer onto apo-ACP. [10] 13 C-NMR confirmed that the 13 Cl abel could be readily observed on derivatised ACPs.A po-BatA was loaded with [ 13 C]-Ac-pantetheine 8,which gave asingle 13 CNMR signal at d = 32.75 ppm ( Figure 3A).…”

Identification and evaluation of quercetin as a potential inhibitor of naphthoate synthase from Enterococcus faecalis

“…aureus . DHNS is a member of the crotonase superfamily, which has extensive roles in primary and secondary metabolism . Thus, taking in account the role of Ef DHNS in the survival of this pathogenic organism, it may be beneficial to determine the structure of Ef DHNS from E .…”