Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

Abstract

The (R)‐2‐hydroxyglutaryl‐CoA dehydratase system from Acidaminococcus fermentans was separated by chromatography of cell‐free extracts on Q‐Sepharose into two components, an activator and the actual dehydratase. The latter enzyme was further purified to homogeneity by chromatography on blue‐Sepharose. It is an iron‐sulfur protein (Mr 210000) consisting of two different polypeptides (α, Mr 55000, and β, Mr 42000) in an α2β2 structure with probably two [4Fe‐4S] centers. After activation this purified enzyme catalysed the dehydration of (R)‐2‐hydroxyglutarate only in the presence of acetyl‐CoA and glutaconate CoA‐transferase, demonstrating that the thiol ester and not the free acid is the substrate of the dehydration. The result led to a modification of the hydroxyglutarate pathway of glutamate fermentation. The activation of the dehydratase by the flow‐through from Q‐Sepharose concentrated by ultrafiltration required NADH, MgCl2, ATP and strict anaerobic conditions. This fraction was designated as A°. Later when the concentration was performed by chromatography on phenyl‐Sepharose, an NADH‐independent form of the activator, designated as A*, was obtained. This enzyme, which required only ATP for activation of the dehydratase, was purified further by affinity chromatography on ATP‐agarose. It contains neither iron nor inorganic sulfur. A*, as well as the activated dehydratase, were irreversibly inactivated by exposure to air within less than 15 min. The activated dehydratase but not A* was also inactivated by 1 mM hydroxylamine or by 0.1 mM 2,4‐dinitrophenol. The (R)‐2‐hydroxyglutaryl‐CoA dehydratase system is closely related the that of (R)‐lactoyl‐CoA dehydratase from Clostridium propionicum as described by R. D. Kuchta and R. H. Abeles [(1985) J. Biol. Chem. 260, 13181–13189].

Nucleotide sequence of the FNR-regulated fumarase gene (fumB) of Escherichia coli K-12

“…Cysteine residues are boxed, the hyphen in P9 denotes an insertion which optimizes the similarity, and the plus sign in P8 denotes an unidentified residue. and the C-terminal segment of fumarase is particularly interesting because two neighboring cysteine residues are conserved, and there is evidence relating disulfide formation in P7 to disruption of the Fe-S cluster of aconitase (22). The existence of such similarities is consistent with the view that the class I fumarases and aconitase are structurally related enzymes.…”

“…The It has previously been suggested that the class I fumarases belong to a family of iron-and sulfur-containing carboxylic acid hydrolyases which includes aconitase, maleate dehydratase, and hydroxyglutaryl-coenzyme A dehydratase (17,22,40). Evidence supporting this view comes from the relatively high instability of these enzymes, their high cysteine contents (1.5% by weight), and the presence of cysteinyl-prolyl sequences.…”

Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin‐dependent Na+ pump glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli

“…Finally GCDD, for which no peptide sequences are known, is also smaller (14 kDa) than the proteins encoded by either of the three OW. Another protein possibly encoded by ORF3 between gcdA and hgdA/ B could be the activator protein which is essential for activity of HGD (Schweiger et al, 1987). Two structures with obvious similarities to a terminator of transcription are located in the 3' region of the hgdB gene.…”

Unusual DAhydrations in anaerobic bacteria: considering ketyls (radical anions) as reactive intermediates in enzymatic reactions

“…1). The oxygen-sensitive dehydratase (HgdAB), contains nonheme iron, inorganic sulfur, riboflavin and FMN [10,11]. The iron and sulfur contents as well a the kind of flavin varies among the enzymes from species (Table 1).…”