Properties and primary structure of a thermostable l-malate dehydrogenase from Archaeoglobus fulgidus
Physicochemical and catalytic properties of thermostable malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 220.127.116.11), isolated from an extreme thermophile, Thermus flavus AT-62, were studied. The enzyme had a molecular weight of 67,000 and consisted of two subunits with an identical molecular weight. The helical content of the enzyme was estimated to be about 25% from the circular dichroism spectrum. The amino acid composition of the thermophilic enzyme was similar to that of its mesophilic counterparts. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) showed that the enzyme contained only one sulfhydryl group per subunit. Substrate inhibition by oxaloacetate was observed. The inhibition decreased with increasing temperature, but was still significant at 60 degrees C. The enzyme was remarkably heat stable, without losing activity after incubation at 90 degrees C for 60 min. The melting temperature of the secondary structure of the enzyme was 96 degrees C.