Substrate specificity of 2-hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum: toward a bio-based production of adipic acid.

Abstract

Expression of six genes from two glutamate fermenting clostridia converted Escherichia coli into a producer of glutaconate from 2-oxoglutarate of the general metabolism (Djurdjevic, I. et al. 2010, Appl. Environ. Microbiol.77, 320-322). The present work examines whether this pathway can also be used to reduce 2-oxoadipate to (R)-2-hydroxyadipic acid and dehydrate its CoA thioester to 2-hexenedioic acid, an unsaturated precursor of the biotechnologically valuable adipic acid (hexanedioic acid). 2-Hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum, the key enzyme of this pathway and a potential radical enzyme, catalyzes the reversible dehydration of (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA. Using a spectrophotometric assay and mass spectrometry, it was found that (R)-2-hydroxyadipoyl-CoA, oxalocrotonyl-CoA, muconyl-CoA, and butynedioyl-CoA, but not 3-methylglutaconyl-CoA, served as alternative substrates. Hydration of butynedioyl-CoA most likely led to 2-oxosuccinyl-CoA, which spontaneously hydrolyzed to oxaloacetate and CoASH. The dehydratase is not specific for the CoA-moiety because (R)-2-hydroxyglutaryl-thioesters of N-acetylcysteamine and pantetheine served as almost equal substrates. Whereas the related 2-hydroxyisocaproyl-CoA dehydratase generated the stable and inhibitory 2,4-pentadienoyl-CoA radical, the analogous allylic ketyl radical could not be detected with muconyl-CoA and 2-hydroxyglutaryl-CoA dehydratase. With the exception of (R)-2-hydroxyglutaryl-CoA, all mono-CoA-thioesters of dicarboxylates used in this study were synthesized with glutaconate CoA-transferase from Acidaminococcus fermentans. The now possible conversion of (R)-2-hydroxyadipate via (R)-2-hydroxyadipoyl-CoA and 2-hexenedioyl-CoA to 2-hexenedioate paves the road for a bio-based production of adipic acid.

• Authors: Anutthaman Parthasarathy, Antonio J Pierik, Jörg Kahnt, Oskar Zelder, Wolfgang Buckel
• Journal: Biochemistry (Biochemistry) Vol. 50 Issue 17 Pg. 3540-50 (May 03 2011) ISSN: 1520-4995 [Electronic] United States
• PMID: 21434666 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Acyl Coenzyme A
• Adipates
• Dicarboxylic Acids
• adipic acid
• alpha-ketoadipic acid
• Alcohol Oxidoreductases
• 2-hydroxyglutarate dehydrogenase
• Coenzyme A-Transferases
• glutaconate CoA-transferase
• 2-hydroxyglutaryl-CoA dehydratase
• Hydro-Lyases

Topics

• Acidaminococcus (enzymology)
• Acyl Coenzyme A (chemistry)
• Adipates (chemistry, metabolism)
• Alcohol Oxidoreductases (chemistry)
• Clostridium symbiosum (enzymology)
• Coenzyme A-Transferases (chemistry)
• Dicarboxylic Acids (chemistry)
• Hydro-Lyases (chemistry)
• Kinetics
• Stereoisomerism
• Substrate Specificity