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.

Reduction of Flavodoxin by Electron Bifurcation and Sodium Ion-dependent Reoxidation by NAD+ Catalyzed by Ferredoxin-NAD+ Reductase (Rnf)

“…After acidification, the CoA thioesters were purified over C18 columns and stored as lyophilized powders at Ϫ80°C (17). The concentration of crotonyl-CoA was calibrated by the NAD ϩ -dependent ␤-oxidation to acetyl-CoA and acetyl phosphate as described for the assay of glutaconyl-CoA decarboxylase (16).…”

Identification and Characterization ofRe-Citrate Synthase in Syntrophus aciditrophicus

“…S. aciditrophicus SB T (ATCC 700169 T ) is available from the McInerney laboratory. Acetyl-CoA was synthesized from CoA and acetic anhydride by an improvement of the method of Simon and Shemin (21,22).…”

Enzyme redesign guided by cancer-derived IDH1 mutations

“…There is great interest in developing biological methods for adipic acid synthesis to lower the cost of input materials, to obviate the need for fossil fuel substrates, and to reduce the release of pollutants compared to standard catalytic methods of synthesis 2–4 . Multiple enzymatic transformations that would support one proposed fully biological method 5 have recently become technically possible 6 . However, the enzymatic conversion of 2-oxoadipate to (R)- 2-hydroxyadipate, a key step in the proposed pathway, remains a major challenge 6 .…”

“…Multiple enzymatic transformations that would support one proposed fully biological method 5 have recently become technically possible 6 . However, the enzymatic conversion of 2-oxoadipate to (R)- 2-hydroxyadipate, a key step in the proposed pathway, remains a major challenge 6 .…”

“…IDH cancer mutants have this desired simple oxidoreductase activity but favor substrates with a 5-carbon backbone, whereas wild-type HIDHs do not have this activity yet act on substrates with a 6-carbon backbone. Here, we applied mutations homologous to those observed in IDHs in cancer to HIDHs to generate a simple oxidoreductase function to convert 2-oxoadipate to (R)- 2-hydroxyadipate 6 , i.e., the desired (R)- 2-hydroxyadipate dehydrogenase (Fig. 1d).…”