D175 Discriminates Between NADH and NADPH in the Coenzyme Binding Site of Lactobacillus delbrueckii subsp. Bulgaricus D-Lactate Dehydrogenase

Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme, Transketolase

“…2A). Site-directed mutagenesis studies on this particular aspartate residue in D-lactate dehydrogenase revealed that this residue discriminates between NADH and NADPH binding (Bernard et al 1995), consistent with its binding to the 2Ј-hydroxyl group of the adenosine ribose of NADH. In some enzymes containing the nucleotidebinding motif-for example, formate dehydrogenasestructural studies have shown that it is the ribose phosphate moieties of the pyridine nucleotide, alone, which bind across the ␤␣␤-fold identified by the motif (Lamzin et al 1992) (Fig.…”

“…Homology between the transketolase sequences shown here is greatest in this N-terminal domain, followed by the middle domain (residues 323-538), which contains a large and extremely well-conserved stretch of amino acids with the consensus sequence (S/T)H(D/C)(S/G)X 3 GX 2 GP(S/ T)(Q/H)X 9 RX 8 (R/Y)PXD where X denotes any amino acid; the residues shown in bold are considered below. Previously, the C-terminal part of this sequence has been identified as being similar to a nucleotide-binding site (Abedinia et al 1992) with the fingerprint sequence GXGXXGX 17-20 D (Bernard et al 1995;Kochhar et al 1992), which has been reported for several enzymes such as malate dehydrogenase (Birktoft et al 1989), glyceraldehyde 3-phosphate dehydrogenase (Skarzynski et al 1987;Murthy et al 1980), alcohol dehydrogenase (Eklund et al 1976), D-and L-lactate dehydrogenases (Bernard et al 1995;Abad-Zapatero et al 1987), and formate dehydrogenase (Lamzin et al 1992). It is thought that this motif is an absolute requirement in order for proteins Table 1.…”

“…4) than in the fingerprint sequence (17)(18)(19)(20). The aspartate residue (Asp503), which was shown to be required for the formation of a ␤␣␤-fold in D-lactate dehydrogenase (Bernard et al 1995), is invariant in all but the recP sequence from S. pneumoniae (Spn). However, correction of an assumed frameshift sequencing error leads to the invariant aspartate and to a threonine that is present in 17 out of the 22 sequences.…”

A New Family ofD-2-Hydroxyacid Dehydrogenases That ComprisesD-Mandelate Dehydrogenases and 2-Ketopantoate Reductases

“…[35][36][37] It is known that the 2-B loop of this domain is responsible for NADH-NADPH discrimination by usual NAD(P)-dependent dehydrogenases. 23,42,43) In the case of E. coli KPR, the guanidino group of Arg31 in the corresponding loop (2-3 loop in the case of the KPR due to an inserted anti-parallel -structure) forms a hydrogen bond with the 2 0 -phosphate of the NADP adenine ribose. 37) In contrast, D-ManDH2 lacks Arg31, and instead has an Asp and Glu at positions 30 and 34 (the numbering of amino acid residues of the enzyme follows that for E. coli KPR) respectively, while the enzyme has the consensus G-X-G-X-X-G motif at the corresponding positions (Fig.…”

“…22) The coenzyme specificities of the enzymes in this family can also be changed by means of protein engineering. [21][22][23][24] In spite of the great variety of their catalytic functions, however, there is no reported enzyme in the D-2-HydDH family that exhibits high catalytic activity toward C3-branched substrates. 17,18,22,25) Enzymes purified from Lactobacillus curvatus, 26) Enterococcus faecalis, 27,28) and also the yeast Rhodotorula graminis 29) are known to catalyze efficiently the conversion between benzoylformate (C 6 H 5 -CO-COO À ) and D-mandelate (C 6 H 5 -CHOH-COO À ), which possess a branched side chain at the C3 position, and are called D-mandelate dehydrogenases (D-ManDHs).…”