2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein

Abstract

Anaerobically prepared cell-free extracts from Fusobacterium nucleatum contain 2-hydroxyglutaryl-CoA dehydratase with a specific activity of 20 nkat mg-1. The enzyme was purified 24-fold to a specific activity of 480 nkat mg-1 by anion exchange chromatography, gel filtration and chromatography on Blue-Sepharose. The activity of the purified enzyme was strictly dependent on the reductant Ti(III)citrate and stimulated 25-fold by 0.15 mM ATP and 5 mM MgCl2. ATP is hydrolysed to ADP during incubation with 2-hydroxyglutaryl-CoA dehydratase in the presence or absence of the substrate. The enzyme is extremely sensitive towards oxygen and is inhibited by 10 microM chloramphenicol, 10 microM 2,4-dinitrophenol or 0.15 mM hydroxylamine. The pure enzyme consists of three subunits alpha (49 kDa), beta (39 kDa) and gamma (24 kDa) in approximately equal amounts. In this respect the enzyme differs from the related 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans and lactyl-CoA dehydratase from Clostridium propionicum both of which are composed of only two subunits with sizes comparable to those of alpha and beta but require an additional protein for activity. The relative molecular mass of the native enzyme of about 100 kDa suggests a trimeric alpha beta gamma-structure. The homogeneous enzyme contains riboflavin (0.5 mol/112 kDa), iron and sulfur (3.5 mol/112 kDa each). Polyclonal antibodies directed against the 2-hydroxyglutaryl-CoA dehydratase from A. fermentans did not crossreact with cell free extracts or purified dehydratase from F. nucleatum. A comparison of the N-terminal amino acid sequences of the dehydratase subunits from A. fermentans and F. nucleatum, however, showed some similarities in the beta-subunits.

Purification and properties of an iron‐sulfur and FAD‐containing 4‐hydroxybutyryl‐CoA dehydratase/vinylacetyl‐CoA3‐2‐isomerase from Clostridium aminobutyricum

“…ATP, 5 mM MgCl, and 0.2 mM of the reducing agent Ti(II1)-citrate, as reported for 2-hydroxyacyl-CoA dehydratases (Schweiger et al, 1987;Klees et al, 1992;Hofmeister and Buckel, 1992), was not observed. Moreover the application of 0.2 mM Ti(II1)citrate to the assay led to a 30% decrease in activity.…”

“…The content of oxidised FAD, iron and labile sulfur led to a new hypothesis for the mechanism of 4-hydroxbutyryl-CoA dehydratase (Fig. 10) analogous to that proposed for lactyl-CoA dehydratase from C. propionicum (Hofmeister and Buckel, 1992) and 2-hydroxyglutarylCoA dehydratase from Fusobacterium nucleatum (Klees et al, 1992). The overall dehydration might proceed in three steps: Firstly, an oxidation of 4-hydroxybutyryl-CoA to 4-hydroxycrotonyl-CoA mediated by the prosthetic group FAD in its oxidised form.…”

“…Dehydratases, which also have to tackle the problem of cleaving an unactivated C-H bond at C-3, are (R)-lactyl-CoA dehydratase from C. propionicum (Kuchta and Abeles, 1985 ;Hofmeister and Buckel, 1992), and (R)-2-hydroxyglutarylCoA dehydratases from A. fermentans (Schweiger et al, 1987) or F: nucleatum (Klees et al, 1992). These enzymes are either composed of two different subunits and require an additional activator protein or of three different subunits.…”

“…On the combined application of all three reagents, however, no enzyme inhibition was determined. Additionally, the alkylating agents iodacetamide and N-ethylmaleimide (10 mM each, 15 min, 22°C) as well as the oxidants chloramphenicol (5 mM), 2,4-dinitrophenol (0.4 mM), hydroxylamine (5 mM) (Klees et al, 1992), 4-nitrophenol (5 mM) and sodium nitrite (10 mM) were without any effect on the enzyme. Furthermore, under the same conditions, the application of 0.5 mM sodium 4-hydroxymercuribenzoate resulted in 50% inhibition.…”

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

“…The dehydratases have to be activated by ATP, Mg 2+ and a reducing agent. In vitro, Ti(III) citrate is applied [11,12,15]; in vivo NADH and probably an additional enzyme serve for this purpose [16]. The activation is catalysed by a polypeptide (HgdC), forming either a third subunit of the dehydratase or an extra separable protein.…”