Thioester Hydrolysis and CC Bond Formation by Carboxymethylproline Synthase from the Crotonase Superfamily

Batchelar, Edward T.; Hamed, Refaat B.; Ducho, Christian; Claridge, Timothy D. W.; Edelmann, Mariola J.; Kessler, Benedikt M.; Schofield, Christopher J.

doi:10.1002/anie.200803906

Cited by 23 publications

(47 citation statements)

References 15 publications

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“…The results imply that negative catalysis, with respect to ethylene formation, has operated during the evolution of 2OG oxygenases and will be useful in protein engineering aimed at optimizing ethylene production. (22). Consistent with the literature (21), ethylene and succinate production depends on L-Arg, with no activity being observed with D-Arg (SI Appendix, Fig.…”

Section: Significancesupporting

confidence: 91%

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

Zhang

Smart

Choi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Ethylene is important in industry and biological signaling. In plants, ethylene is produced by oxidation of 1-aminocyclopropane-1-carboxylic acid, as catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase. Bacteria catalyze ethylene production, but via the fourelectron oxidation of 2-oxoglutarate to give ethylene in an argininedependent reaction. Crystallographic and biochemical studies on the Pseudomonas syringae ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate-derived intermediate occurs to give ethylene and carbon dioxide. Crystallographic and quantum chemical studies reveal that fragmentation to give ethylene is promoted by binding of L-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation.ethylene-forming enzyme | 2-oxoglutarate-dependent oxygenases | hydroxylase | plant development | oxidoreductase E thylene is of industrial importance and is a vital signaling molecule in plants, where it has roles in germination, senescence, and stress responses (1). Commercial manipulation of the natural ethylene response is agriculturally important in controlling fruit ripening (2). In higher plants, ethylene is produced from methionine, via oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) in an unusual reaction catalyzed by the Fe(II)-dependent ACC oxidase (ACCO) (3, 4), which is part of the 2-oxoglutarate (2OG)-dependent oxygenase superfamily, although it does not use a 2OG cosubstrate (Fig. 1A) (5-7). Ethylene is also produced in some microorganisms by oxidation of 2-oxo-4-methylthiobutyric acid in a reaction not directly enzyme catalyzed (8,9).In work aimed at producing industrial ethylene by biocatalysis, Pseudomonas strains, including plant pathogens, were shown to produce large amounts of ethylene (10)(11)(12)(13)(14). Bacteria engineered to produce ethylene using the Pseudomonas syringae pv. phaseolicola ethylene-forming enzyme (PsEFE) have been developed to ripen fruit as an alternative to the use of synthetic ethylene (15, 16). Ethylene-forming enzymes are being explored for biocatalysis in cyanobacteria (17-19). PsEFE-catalyzed ethylene production is 2OG-dependent and is stimulated by the addition of L-arginine (L-Arg), which is also converted by PsEFE into pyrroline-5-carboxylate (P5C; Fig. 1B) (13, 20). In contrast to the consensus 2OG oxygenase mechanism, which involves sequential binding of 2OG, substrate, and then oxygen, an unprecedented "dual circuit" mechanism is proposed for PsEFE (13).We describe biochemical, structural, and modeling studies supporting a branched mechanistic pathway for PsEFE that can lead either to ethylene via oxidative fragmentation of 2OG or to succinate via a more typical 2OG oxygenase reaction, which sometimes results in P5C formation (Fig....

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Section: Significancesupporting

confidence: 91%

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

Zhang

Smart

Choi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Regiospecific methylation of polyketide products using organic semisynthesis is challenging. [4] In further support of the proposed Route 1, attack of the enolate on the upstream acyl-KS should occur more readily with the less sterically hindered acetyl enolate, than the more sterically hindered isobutyryl enolate of Route 2. [2] Type I polyketide synthases catalyze Claisen condensations and tailoring reactions in an assembly line fashion to elaborate a remarkably diverse collection of secondary metabolites, many of which have medicinal and industrial applications.…”

mentioning

confidence: 84%

“…[5] Dimethyl-malonyl groups are also naturally added to sugar residues in the biosynthesis of cervimycin, [6] and have been proposed as a source of isobutyryl groups in polyketide synthesis, but the latter has not been conclusively demonstrated. When provided in vitro to carboxymethylproline synthase (CarB), a member of the crotonase superfamily, a dimethylmalonyl moiety has been shown to be a source of a nucleophilic enolate for CÀC bond-forming reactions, despite its steric bulk.…”

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confidence: 99%

Divergent Mechanistic Routes for the Formation of gem‐Dimethyl Groups in the Biosynthesis of Complex Polyketides

et al. 2015

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The gem-dimethyl groups in polyketide-derived natural products add steric bulk and, accordingly, lend increased stability to medicinal compounds, however, our ability to rationally incorporate this functional group in modified natural products is limited. In order to characterize the mechanism of gem-dimethyl group formation, with a goal toward engineering of novel compounds containing this moiety, the gem-dimethyl group producing polyketide synthase (PKS) modules of yersiniabactin and epothilone were characterized using mass spectrometry. The work demonstrated, contrary to the canonical understanding of reaction order in PKSs, that methylation can precede condensation in gem-dimethyl group producing PKS modules. Experiments showed that both PKSs are able to use dimethylmalonyl acyl carrier protein (ACP) as an extender unit. Interestingly, for epothilone module 8, use of dimethylmalonyl-ACP appeared to be the sole route to form a gem-dimethylated product, while the yersiniabactin PKS could methylate before or after ketosynthase condensation.

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“…Elimination of CoA produces a ketene intermediate, which is followed by the addition of a hydroxide to generate the acid product. Finally, hydrolysis of the CoA ester by the enzyme CarB most likely proceeds through the attack of the carbonyl carbon by a water molecule activated by an active site glutamate side chain [9].…”

Section: Resultsmentioning

confidence: 99%

“…Of special interest, the two acidic residues that are important for the catalysis of ECH (Glu144 and Glu164) are also conserved in DmdD (Glu121 and Glu141). Enzymes in the crotonase superfamily are mechanistically diverse and catalyze many different types of reactions on CoA esters [5], including hydration [6], hydrolysis [7], [8], [9], [10], isomerization [11], dehalogenation [12], decarboxylation [13], and others.…”

Section: Introductionmentioning

confidence: 99%

Crystal Structure of DmdD, a Crotonase Superfamily Enzyme That Catalyzes the Hydration and Hydrolysis of Methylthioacryloyl-CoA

et al. 2013

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Dimethyl-sulphoniopropionate (DMSP) is produced in abundance by marine phytoplankton, and the catabolism of this compound is an important source of carbon and reduced sulfur for marine bacteria and other organisms. The enzyme DmdD catalyzes the last step in the methanethiol (MeSH) pathway of DMSP catabolism. DmdD is a member of the crotonase superfamily of enzymes, and it catalyzes both the hydration and the hydrolysis of methylthioacryloyl-CoA (MTA-CoA), converting it to acetaldehyde, CO2, MeSH, and CoA. We report here the crystal structure of Ruegeria pomeroyi DmdD free enzyme at 1.5 Å resolution and the structures of the E121A mutant in complex with MTA-CoA and 3-methylmercaptopropionate-CoA (MMPA-CoA) at 1.8 Å resolution. DmdD is a hexamer, composed of a dimer of trimers where the three monomers of each trimer are related by a crystallographic 3-fold axis. The overall structure of this hexamer is similar to those of canonical crotonases. However, the C-terminal loops of DmdD in one of the trimers assume a different conformation and contribute to CoA binding in the active site of a neighboring monomer of the trimer, while these loops in the second trimer are disordered. MTA-CoA is bound deep in the active site in the first trimer, but shows a 1.5 Å shift in its position in the second trimer. MMPA-CoA has a similar binding mode to MTA-CoA in the first trimer. MMPA-CoA cannot be hydrated and is only hydrolyzed slowly by DmdD. Replacement of the sulfur atom in MMPA-CoA with a methylene group abolishes hydrolysis, suggesting that the unique property of the substrate is a major determinant of the hydrolysis activity of DmdD.

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Thioester Hydrolysis and CC Bond Formation by Carboxymethylproline Synthase from the Crotonase Superfamily

Cited by 23 publications

References 15 publications

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

Divergent Mechanistic Routes for the Formation of gem‐Dimethyl Groups in the Biosynthesis of Complex Polyketides

Crystal Structure of DmdD, a Crotonase Superfamily Enzyme That Catalyzes the Hydration and Hydrolysis of Methylthioacryloyl-CoA

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