Crystal structure of the MJ0490 gene product of the hyperthermophilic archaebacterium Methanococcus jannaschii, a novel member of the lactate/malate family of dehydrogenases

MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was cla ssified initially as coding for a putative L-lactate dehydrogenase (LDH). I t has been re-classified as a malate dehydrogenase (MDH) gene, because it s hows significant sequence similarity to MT0188, MDH II from Methanobacteriu m thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structu re in the orthorhombic crystal at 1.9 Angstrom resolution and a "tetrameric " structure in the tetragonal crystal at 2.8 Angstrom. These structures sha re a similar subunit fold with other LDHs and MDHs. The tetrameric structur e resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to t he Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during p rotein purification and crystallization. The preference of NADP(H) over NAD (H) has been supported by activity assays. The cofactor preference is expla ined by the presence of a glycine residue in the cofactor binding pocket (G ly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydro gen bonds between the oxygen atoms of the monophosphate group and the ribos e sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is c onserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ049 0 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (r eduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibi ting a relaxed substrate and cofactor specificities in NADP(H) and NAD(H)-d ependent malate and lactate dehydrogenase reactions. (C) 2001 Academic Pres s.