PMID:9931298

Citation	Tetaud, E, Hanau, S, Wells, JM, Le Page, RW, Adams, MJ, Arkison, S and Barrett, MP (1999) 6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme. Biochem. J. 338 ( Pt 1):55-60
Abstract	A gene encoding 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1. 44) was identified from the homofermentative lactic acid bacterium Lactococcus lactis, by complementation of Escherichia coli mutants. The cloned gene was then expressed to high levels in E. coli and the protein purified for kinetic analysis. The enzyme had a Km for 6-phosphogluconate of 15.4+/-1.4 microM and for NADP of 1.9+/-0.2 microM at pH 7.5. Sequence comparison of the L. lactis 6-PGDH with the corresponding enzyme derived from the pathogenic protozoan Trypanosoma brucei and sheep liver revealed the substrate-binding residues to be identical in all three species, although the three coenzyme-binding pockets differed slightly. A totally conserved arginine residue (Arg-447), believed to bind the 6-phosphate of substrate, was mutated to lysine, aspartate, alanine or tryptophan. In each case enzyme activity was lost, confirming an essential role for this residue on activity. A second arginine (Arg-34), believed to be critical in binding the 2'-phosphate of cofactor NADP+, was mutated to a tyrosine residue, as found in one atypical isoform of the enzyme in Bacillus subtilis. This alteration led to decrease in affinity 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 contains an arginine (Arg-34) in this position, suggesting that B. subtilis has two 6-PGDHs with different coenzyme specificities.
Links	PubMed PMC1220024
Keywords	Amino Acid Sequence; Animals; Arginine/genetics; Arginine/physiology; Bacillus subtilis/enzymology; Binding Sites/genetics; Escherichia coli/genetics; Genes, Bacterial; Genetic Complementation Test; Isoenzymes/metabolism; Kinetics; Lactococcus lactis/enzymology; Lactococcus lactis/genetics; Molecular Sequence Data; NAD/metabolism; NADP/metabolism; Phosphogluconate Dehydrogenase/genetics; Phosphogluconate Dehydrogenase/metabolism; Sheep; Substrate Specificity/genetics; Trypanosoma brucei brucei/enzymology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
LACLM:6PGD		GO:0006525: arginine metabolic process	ECO:0000315:		P		As shown on page 57, "The role of the L. lactis 6-PGDH Arg-447 in binding of substrate" paragraph of the associated PMID:"Despite the fact that the mutated [6-PGDH] enzymes were all soluble... none had any activity whatsoever... confirming that this [Arginine] residue plays a critical role in activity."	complete CACAO 9641
BIFAP:K4IP48		GO:0004616: phosphogluconate dehydrogenase (decarboxylating) activity	ECO:0000314:		F		Table 2	complete CACAO 9572

References

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