6-phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme

A gene encoding 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1.44) was identified from the homofermentative lactic acid bacterium Lactococcus lact is, by complementation of Escherichia coli mutants. The cloned gene was the n expressed to high levels in E. coli and the protein purified for kinetic analysis. The enzyme had a K-m for 6-phosphogluconate of 15.4 +/- 1.4 mu M and for NADP of 1.9 +/- 0.2 mu M at pH 7.5. Sequence comparison of the L. l actis 6-PGDH with the corresponding enzyme derived from the pathogenic prot ozoan Trypanosoma brucei and sheep liver revealed the substrate-binding res idues to be identical in all three species, although the three coenzyme-bin ding pockets differed slightly. A totally conserved arginine residue (Arg-4 47), believed to bind the 6-phosphate of substrate, was mutated to lysine, aspartate, alanine or tryptophan. In each case enzyme activity was lost, co nfirming an essential role for this residue on activity. A second arginine (Arg-34), believed to be critical in binding the 2'-phosphate of cofactor N ADP(+), was mutated to a tyrosine residue, as found in one atypical isoform of the enzyme in Bacillus subtilis. This alteration led to decrease in aff inity for NADP(+) of nearly three orders of magnitude. A second 6-PGDH gene has been identified from the genome of B. subtilis. This second isoform co ntains an arginine (Arg-34) in this position. suggesting that B. subtilis h as two 6-PGDHs with different coenzyme specificities.