d -(–)-Tartrate Dehydratase of Rhodopseudomonas sphaeroides : Purification, Characterization, and Application to Enzymatic Determination of d -(–)-Tartrate

Abstract

An isolate of Rhodopseudomonas sphaeroides was capable of growing phototrophically and chemotrophically (μ = 0.15 h −1 for either condition) with d -(–)-tartrate as the carbon source. A d -(–)-tartrate dehydratase, ( d -(–)-tartrate hydrolyase, EC 4.1.2.70) was induced in the presence of d -(–)-tartrate. The enzyme was purified 30-fold from cell extracts of R. sphaeroides to a specific activity of 7.5 U/mg of protein and was subsequently crystallized in the presence of 1 M KCl. The enzyme was homogeneous upon analytical electrophoresis in 5% polyacrylamide gels and by criteria of ultracentrifugation. The native enzyme had a molecular weight of 158,000 ± 1,000 as determined by gel filtration and ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 39,500 ± 500, indicating that d -(–)-tartrate dehydratase was a tetramer. The isoelectric point of the native enzyme was at pH 5.5. The enzyme catalyzed irreversibly the conversion of d -(–)-tartrate to oxaloacetate and water, and the turnover number was calculated to be 1,185. The reaction followed Michaelis-Menten kinetics, and a K m value of 1.8 × 10 −4 M was determined. d -(–)-Tartrate dehydratase required Mg 2+ for activity. The pH optimum was within a range from 6.2 to 7.2, and the activation energy of the reaction (Δ H 0 ) was 63.2 kJ/mol. The enzyme was specific for d -(–)-tartrate; it did not react with l -(+)-tartrate, meso -tartrate, and other hydroxycarboxylic acids. d -(–)-Tartrate dehydratase was strongly inhibited by meso -tartrate (50% at 0.6 mM). l -(+)-Tartrate and a variety of hydroxycarboxylic acids caused 50% inhibition at concentrations of >30 mM.

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

“…However, there is no convincing evidence that those clusters are involved in the chemical mechanism of the dehydration. Moreover, some closely related dehydratases such as fumarase from pig heart and D-( -)tartrate dehydratase from Rhodopseudornonus sphaeroides [35] do not contain iron and sulfur; see also [36].…”

Microbial Production of 2-Oxobutyric Acid from Crotonic Acid

“…However, the largest amount of OBA was accumulated at 25°C in 24 hr. These results may be due to the loss of the activity over 19,20) are• known to require divalent cations. The effects of divalent cations on OBA production were examined.…”

Adaptation of Rhodopseudomonas sphaeroides to Growth on d -(—)-Tartrate and Large-Scale Production of a Constitutive d -(—)-Tartrate Dehydratase During Growth on dl -Malate

“…Growth on D-(-)-tartrate has been reported for enterobacteria (1,4,24), pseudomonads (7,9,(19)(20)(21), and Rhodopseudomonas sphaeroides (18). For R. sphaeroides (18) and two Pseudomonas strains (19), it has been shown that this compound is metabolized by highly specific D-(-)-tartrate dehydratases. One of these enzymes has been used in a quantitative microassay for D-(-)-tartrate (18).…”

“…For R. sphaeroides (18) and two Pseudomonas strains (19), it has been shown that this compound is metabolized by highly specific D-(-)-tartrate dehydratases. One of these enzymes has been used in a quantitative microassay for D-(-)-tartrate (18). D-(-)-Tartaric acid is a component of racemic tartaric acid, from which it can be obtained by selective degradation of L-(+)-tartaric acid by microorganisms (12).…”

“…It has recently been shown that R. sphaeroides Si-4 exhibits an extended lag phase of approximately 3 days when inocula grown on media containing succinate, malate or L-(+)-tartrate are transferred to a medium containing D-(-)-tartrate. After the log phase, the organism grew exponentially and either phototrophically or chemotrophically, with rates of 0.15 h-1 (18).…”