title: 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: Fachbereich Biologie, Philipps Universität, Marburg, Germany.
authors: Anutthaman
authors: A
authors: Parthasarathy
authors: Antonio J
authors: AJ
authors: Pierik
authors: Jörg
authors: J
authors: Kahnt
authors: Oskar
authors: O
authors: Zelder
authors: Wolfgang
authors: W
authors: Buckel
mesh_terms: D045850:Acidaminococcus / Q000201:enzymology; D000214:Acyl Coenzyme A / Q000737:chemistry; D000272:Adipates / Q000737:chemistry / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000737:chemistry; D048009:Clostridium symbiosum / Q000201:enzymology*; D015256:Coenzyme A-Transferases / Q000737:chemistry; D003998:Dicarboxylic Acids / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry*; D007700:Kinetics; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D000272:Adipates; D003998:Dicarboxylic Acids; C029900:adipic acid; C001918:alpha-ketoadipic acid; D000429:Alcohol Oxidoreductases; C023858:2-hydroxyglutarate dehydrogenase; D015256:Coenzyme A-Transferases; C032430:glutaconate CoA-transferase; C054464:2-hydroxyglutaryl-CoA dehydratase; D006836:Hydro-Lyases