methylmalonyl-CoA Mutase Reaction Research Articles

An erythromycin process improvement using the diethyl methylmalonate-responsive (Dmr) phenotype of the Saccharopolyspora erythraea mutB strain

The Saccharopolyspora erythraea mutB knockout strain, FL2281, having a block in the methylmalonyl-CoA mutase reaction, was found to carry a diethyl methylmalonate-responsive (Dmr) phenotype in an oil-based fermentation medium. The Dmr phenotype confers the ability to increase erythromycin A (erythromycin) production from 250-300% when the oil-based medium is supplemented with 15 mM levels of this solvent. Lower concentrations of the solvent stimulated proportionately less erythromycin production, while higher concentrations had no additional benefit. Although the mutB strain is phenotypically a low-level erythromycin producer, diethyl methylmalonate supplementation allowed it to produce up to 30% more erythromycin than the wild-type (control) strain-a strain that does not show the Dmr phenotype. The Dmr phenotype represents a new class of strain improvement phenotype. A theory to explain the biochemical mechanism for the Dmr phenotype is proposed. Other phenotypes found to be associated with the mutB knockout were a growth defect and hyper-pigmentation, both of which were restored to normal by exposure to diethyl methylmalonate. Furthermore, mutB fermentations did not significantly metabolize soybean oil in the presence of diethyl methylmalonate. Finally, a novel method is proposed for the isolation of additional mutants with the Dmr phenotype.

Assessing the Reversibility of the Anaplerotic Reactions of the Propionyl-CoA Pathway in Heart and Liver

While a number of studies underline the importance of anaplerotic pathways for hepatic biosynthetic functions and cardiac contractile activity, much remains to be learned about the sites and regulation of anaplerosis in these tissues. As part of a study on the regulation of anaplerosis from propionyl-CoA precursors in rat livers and hearts, we investigated the degree of reversibility of the reactions of the propionyl-CoA pathway. Label was introduced into the pathway via NaH13CO3, [U-13C3]propionate, or [U-13C3]lactate + [U-13C3]pyruvate, under various concentrations of propionate. The mass isotopomer distributions of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA revealed that, in intact livers and hearts, (i) the propionyl-CoA carboxylase reaction is slightly reversible only at low propionyl-CoA flux, (ii) the methylmalonyl-CoA racemase reaction keeps the methylmalonyl-CoA enantiomers in isotopic equilibrium under all conditions tested, and (iii) the methylmalonyl-CoA mutase reaction is reversible, but its reversibility decreases as the flow of propionyl-CoA increases. The thermodynamic dis-equilibrium of the combined reactions of the propionyl-CoA pathway explains the effectiveness of anaplerosis from propionyl-CoA precursors such as heptanoate.

Theoretical evaluation of the hydrogen kinetic isotope effect on the first step of the methylmalonyl-CoA mutase reaction

We have calculated hydrogen kinetic isotope effects (KIEs) for the first step of the methylmalonyl-CoA mutase reaction, including multidimensional tunneling correction at the zero curvature (ZCT) level, and compared them with the experimental values. Both alternative mechanisms of this step, concerted and stepwise, can be accommodated. It turned out to be essential to include Arg207 hydrogen-bonded to the reactant in the mechanism predicting simultaneous breaking of the Co–C bond of AdoCbl and hydrogen atom transfer. The consequence of the stepwise mechanism is a much larger facilitation of the homolytic dissociation of the carbon–cobalt bond by the enzyme than currently appreciated; our results suggest lowering of the activation energy by about 23 kcal mol−1. We have also shown that large hydrogen KIEs of tunneling origin do not necessarily break the Swain–Schaad equation. Furthermore, when this equation does not hold, the exponent may be smaller in the presence of tunneling than it is at the semi-classical limit, indicating that nonclassical behavior may be a more common phenomenon than expected.

Evidence for a 1,2 Shift of a Hydrogen Atom in a Radical Intermediate of the Methylmalonyl-CoA Mutase Reaction

An excellent substrate of methylmalonyl-CoA mutase, methylmalonyl-carba-(dethia) coenzyme A (methylmalonyl-CH2-CoA), was synthesized by a chemoenzymatic method and its α-proton was exchanged with deuterium by long-term incubation in deuterium oxide at pH 6.9. After addition of highly purified epimerase-free methylmalonyl-CoA mutase the enzymatic rearrangement was monitored by 1H NMR spectroscopy. Already in the initial phases of the reaction only 72% of the produced succinyl-CH2-CoA was monodeuterated, while unlabeled and geminally dideuterated species, 14% of each, were also formed. After the addition of more enzyme the equilibrium (methylmalonyl-CoA:succinyl-CoA = 1:20) was quickly established, while the proportion of unlabeled succinyl-CH2-CoA rose to 30% and the geminally dideuterated species were slowly transformed to vicinally dideuterated ones. After 19 h of incubation the ratio of the unlabeled, monodeuterated, and dideuterated species was roughly 1:1:1 while no appreciable deuterium incorporation from the solvent occurred. The unexpected disproportionation of deuterium can be best explained by a 1,2 shift of a hydrogen atom in the succinyl-CH2-CoA radical intermediate competing with the hydrogen transfer from 5′-deoxyadenosine. A precedence for such a hydrogen shift in a radical was previously observed only in the mass spectrometer and was supported by ab initio calculations.

The Enzymic Interconversion of Isobutyryl and n‐Butyrylcarba(dethia)‐Coenzyme A: A Coenzyme‐B12‐dependent Carbon Skeleton Rearrangement

The synthetic carba(dethia) analogues of coenzyme A contain a —CO—CH2—instead of a —CO—S— group and are therefore hydrolytically stable. The isobutyrylcarba(dethia) coenzyme A 1, whose synthesis is reported here, is isomerized to the n-butyryl derivative 2 by a cell extract from Streptomyces cinnamonensis (monitored by 1H NMR). This new, coenzyme B12-dependent structural rearrangement is similar in many ways to the long-known methylmalonyl-CoA mutase reaction.

Further studies on the biosynthesis of chlorothricin.

Feeding experiments with [U-13C3]- and (2R)-[1-2H2]glycerol showed that glycerol is incorporated intact into carbon atoms 22, 23 and 24 of the aglycone of chlorothricin. C-1 of glycerol gives rise to C-22 with retention of one atom of deuterium, which occupies the H-22R position. A mechanism for the assembly of the aglycone is proposed which invokes phosphoenolpyruvate as the direct precursor of the 3-carbon moiety and a Baeyer-Villiger oxidation as the mode of formation of the macrocyclic lactone functionality. A feeding experiment with [1,2-13C2]succinate suggests that the propionate units of the aglycone polyketide are formed entirely via the methylmalonyl-CoA mutase reaction. The formation of the two 2,6-dideoxy-D-rhamnose moieties of chlorothricin from glucose was shown to involve replacement of the 2-hydroxyl group of the sugar by hydrogen with inversion of configuration at C-2. This contrasts with the retention stereochemistry observed earlier for the analogous formation of the 2,6-dideoxyhexose moiety of the antibiotic granaticin.

ChemInform Abstract: FREE RADICAL REARRANGEMENT INVOLVING THE 1,2‐MIGRATION OF A THIOESTER GROUP. MODEL FOR THE COENZYME B12‐DEPENDENT METHYLMALONYL‐COA MUTASE REACTION

Chemischer InformationsdienstVolume 16, Issue 17 Natural Products ChemInform Abstract: FREE RADICAL REARRANGEMENT INVOLVING THE 1,2-MIGRATION OF A THIOESTER GROUP. MODEL FOR THE COENZYME B12-DEPENDENT METHYLMALONYL-COA MUTASE REACTION S. WOLLOWITZ, S. WOLLOWITZSearch for more papers by this authorJ. HALPERN, J. HALPERNSearch for more papers by this author S. WOLLOWITZ, S. WOLLOWITZSearch for more papers by this authorJ. HALPERN, J. HALPERNSearch for more papers by this author First published: April 30, 1985 https://doi.org/10.1002/chin.198517311AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume16, Issue17April 30, 1985 RelatedInformation

Additions and Corrections - Free Radical Rearrangement Involving the 1,2-Migration of a Thioester Group. Model for the Coenzyme B12 Dependent Methylmalonyl-CoA Mutase Reaction

Additions and Corrections - Free Radical Rearrangement Involving the 1,2-Migration of a Thioester Group. Model for the Coenzyme B₁₂ Dependent Methylmalonyl-CoA Mutase Reaction

Free radical rearrangement involving the 1,2-migration of a thioester group. Model for the coenzyme B12-dependent methylmalonyl-CoA mutase reaction

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTFree radical rearrangement involving the 1,2-migration of a thioester group. Model for the coenzyme B12-dependent methylmalonyl-CoA mutase reactionSusan Wollowitz and Jack HalpernCite this: J. Am. Chem. Soc. 1984, 106, 26, 8319–8321Publication Date (Print):December 1, 1984Publication History Published online1 May 2002Published inissue 1 December 1984https://doi.org/10.1021/ja00338a066RIGHTS & PERMISSIONSArticle Views336Altmetric-Citations58LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (391 KB) Get e-Alerts Get e-Alerts

Mechanism of coenzyme-B12-dependent molecular rearrangements. Evidence for a radical-like process in a model reaction for methylmalonyl–CoA mutase

Mechanistic probes of a model system for the coenzyme-B12-dependent methylmalonyl–CoA mutase reaction have suggested (i) that the C–Co bond in the alkylcobalamin intermediate is capable of both carbanionic and homolytic cleavage, and (ii) that the skeletal rearrangement most probably takes place at the radical stage.

Investigation of the mechanism of the methylmalonyl-CoA mutase reaction with the substrate analogue: ethylmalonyl-CoA.

1. Ethylmalonyl-CoA was found to be a substrate for methylmalonyl-CoA mutase from Propionibacterium shermanii, the product being mainly (2R)-methylsuccinyl-CoA along with some (2S)-diastereoisomer. 2. The relevant 1H-nuclear magnetic resonance signals of methylsuccinic acid and of its dimethyl ester were assigned to the diastereotopic methylene hydrogens using sterospecifically dideuterated specimens of known configuration. 3. [2(-2)H1]Ethylmalonyl-CoA was converted by methylmalonyl-CoA mutase in 2H2O mainly to (2R, 3S)-[3(-2)H1]methylsuccinyl-CoA. No dideuterated product was observed. 4. Starting from (1R)-[1(-2)H1]-ethathanol, (1S)-[1(-2)H1]ethanol and [2H6] ethanol the following deuterated specimens of ethylmalonic acid were synthesised and characterised: (3S)-[3(-2)H1], (3R)-[3(-2)H1] and [3(-2)H2, 4(-2)H3], respectively. 5. Conversion of (3S)-[3(-2)H1]-ethylmalonyl-CoA (70% 2H1 and 2% 2H2 species) on the mutase in water afforded mainly (2R)-[2(-2)H1]methylsuccinyl-CoA along with some (2S)-diastereoisomer. No deuterium loss was observed. 6. Methylmalonyl-CoA mutase converted (3R)-[3(-2)H1]ethylmalonyl-CoA (81% 2H1 and 2% 2H2 species) to the following methylsuccinyl-CoA species: 33% [3(-2)H1], the deuterium being in the threo position with respect to the methyl group; 21% [2(-2)H1]; 46% unlabelled. The ratio of the species with (2R) and (2S) configuration was about 60:40. 7. Reaction of [3(-2)H2, 4(-2)H3]ethylmalonyl-CoA (94.5% [2H5] species) with the mutase gave the following labelled methylsuccinyl-CoA species:53.4% [methyl-2H3, 2(-2)H1, 3(-2)H1], the 3-deuterium being in the threo position with respect to the methyl group; 37.6% [methyl-2H3, 2(-2)H1]; 5% [methyl(-2)H3, 2(-2)H1, 2(-2)H1, 3(-2)H1] the 3-deuterium being in erythro position with respect to the methyl group; 4% [methyl(-2)H3, 3(-2)H1]. The ratio of the species with (2R) and (2S) configuration was about 70:30. 8. Implications of these findings for the mechanism of the rearrangements catalysed by coenzyme B12 are discussed.

An Effect of Vitamin B12 Deficiency in Tissue Culture

Rat glial cells were grown in tissue culture in vitamin B12-deficient medium. As the intracellular level of coenzyme B12 decreased, the cells made increasing amounts of two unusual fatty acids. The fatty acids were unbranched acids with 15 and 17 carbon atoms and arose as the cells progressively lost the ability to metabolize propionic acid through the B12-requiring methylmalonyl-CoA mutase reaction. The levels of the 15- and 17-carbon acids returned to normal when the cultures were supplemented with vitamin B12. The possible importance of similar fatty acid changes in human vitamin B12 deficiency and in disorders of 3- and 4- carbon acid metabolism is speculated.

The absolute configuration of methylmalonyl CoA and stereochemistry of the methylmalonyl CoA mutase reaction

The absolute configuration of methylmalonyl CoA and stereochemistry of the methylmalonyl CoA mutase reaction