Phylogenetic relationships and biochemical properties of the duplicated cytosolic and mitochondrial isoforms of malate dehydrogenase from a teleost fish, Sphyraena idiastes.
Unlike birds and mammals, teleost fish express two paralogous isoforms (paralogues) of cytosolic malate dehydrogenase (cMDH; EC 1.1.1.37; NAD+: malate oxidoreductase) whose evolutionary relationships to the single cMDH of tetrapods are unknown. We sequenced complementary DNAs for both cMDHs and the mitochondrial isoform (mMDH) of the fish Sphyraena idiastes (south temperate barracuda) and compared the sequences, kinetic properties, and thermal stabilities of the three isoforms with those of mammalian orthologues. Both fish cMDHs comprise 333 residues and have subunit masses of approximately 36 kDa. One cytosolic isoform, cMDH-S, was significantly more heat-stable than either the other cMDH (cMDH-L) or mMDH. In contradiction to the generally accepted model of vertebrate cMDH evolution, our phylogenetic analysis indicates that the duplication of the fish cytosolic paralogues occurred after the divergence of the lineages leading to teleosts and tetrapods. cMDH-L and cMDH-S differed in optimal concentrations of substrates and cofactors and apparent Michaelis-Menten constants, suggesting that the two paralogues may play distinct physiological roles. Differences in intrinsic thermal stability among MDH paralogues may reflect different degrees of stabilization in vivo by extrinsic stabilizers, notably protein concentration in the case of mMDH. Thermal stabilities of porcine mMDH and cMDH-L, but not cMDH-S, were significantly increased when denaturation was measured at a high protein (bovine serum albumin; BSA) concentration, but the BSA-induced stabilization reduced the catalytic activity.
Lin
Jen-Jen
JJ
Hopkins Marine Station, Department of Biological Sciences, Stanford University, Pacific Grove, CA 93950-3094 USA.
Yang
Tzung-Horng
TH
Wahlstrand
Benjamin D
BD
Fields
Peter A
PA
Somero
George N
GN
D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D005399:Fishes / Q000145:classification / Q000235:genetics*; D007527:Isoenzymes / Q000145:classification / Q000235:genetics; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000145:classification / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010802:Phylogeny; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013020:South America; D013696:Temperature
D007527:Isoenzymes; D008291:Malate Dehydrogenase