Enzymic formation of D-malate.

Tubbs & Greville (1961) showed that D-2hydroxy acid dehydrogenase (EC 1.1.2.4), present in liver and kidney in several animal species, catalyses the oxidation of D-lactate, D-malate and various straight-chain homologues. Similar NADindependent D-lactate dehydrogenases have been isolated from micro-organisms (Haugaard, 1959; de Ley & Schel, 1959; Snoswell, 1959; Nygaard, 1960). However, the metabolic significance of the enzyme, or enzymes, has still to be determined since this involves an assessment of the roles of those reactions that result in the formation of D-lactate, D-malate and other D-2-hydroxy acids. When maleate is formed in metabolism it is usually isomerized to fumarate before hydration to give L-malate. This reaction was shown to occur when nicotinic acid is degraded by a strain ofPseudomona8 fluore8cen8 (Behrman & Stanier, 1957) and a similar isomerase is induced when Ataligene8 faecati8 is exposed to malonate (Takamura, Nakatani, Soejima & Aoyama, 1968). The maleyl configuration arises from the oxidative cleavage of the benzene nucleus of homogentisate (Edwards & Knox, 1956) and gentisate (Lack, 1959) and in these cases maleylacetoacetate and maleylpyruvate respectively are isomerized to tran8 forms before hydrolysis, so that water is added to fumarate and not to maleate. In the present work we have used a different species of P8eudomonaa from that of Lack (1959) and have extracted enzymes that convert gentisate or maleate into D-malate, apparently by the reaction sequence, which does not involve an isomerization, shown in Scheme 1. The organism used, P8eudomona8 N.C.I.B. 9867, was grown with forced aeration at 30° in a medium that contained (per 1.): Na2HPO4, 4-33g.; KH2PO4, 2-65g.; NH4CI, 2-0g.; MgSO4,7H20, 0-4g.; m-cresol, 0-3g. Cell extracts, obtained by ultrasonic treatment as described by Dagley & Gibson (1965), were partially purified to give two types of enzyme preparations, which we refer to as 'dialysed' extracts or 'Sephadex-treated' extracts. A dialysed extract, which oxidized gentisate to malate and pyruvate in the presence of Fe2+ ions, was obtained when a crude cell extract was brought to 70% saturation with (NH4)2504, the precipitate was dissolved in a volume of 0-05m-Na2HPO4-KH2PO4 buffer, pH7, equal to that ofthe original extract, and the solution was dialysed for 3hr. against two changes of the same buffer. Sephadex-treated extracts contained gentisate oxygenase but lacked the hydrolase for maleylpyruvate; accordingly they were used to prepare the last-named compound. The protein precipitated from a crude extract between 40% and 70% saturation with (NH4)2SO4 was dissolved in 20ml. of 0-1M-Na2HPO4-KH2PO4 buffer, pH7, containing 5% (v/v) of saturated (NH4)2S04. This solution (19mg. of protein/ml.; pH 6-8) was applied to a column (95cm. x 5-5cm.) of Sephadex G-150 and eluted with the same buffer (flow rate 39ml./hr.). The fractions that contained gentisate oxygenase were pooled, precipitated with (NH4)2SO4 and redissolved in a smaller volume of the buffer. When 5 ,umoles of gentisate were metabolized by dialysed extract in a Warburg respirometer, 5,umoles of 02 were consumed and 5-2,umoles of pyruvate were formed, as determined by the method of Friedemann & Haugen (1943). The second product of the reaction was identified as D-malate by the following experiment. A 250ml. conical flask, which contained 50ml. of dialysed extract, 50ml. of 0-1M-Na2HPO4-KH2PO4 buffer, pH7, and 2ml. of 0-2M-FeSO4 was agitated at 300 on a rotatory shaker. Sodium gentisate was added until the reaction ceased, as determined by measurements performed with portions of the reaction mixture by