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

Tetaud, E., Hanau, S., Wekks, J. M., Le Page, R. W.F., Adams, M. J., Arkison, S. and Barrett, M. P. (1999) 6-phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme. Biochemical Journal, 338(1), pp. 55-60. (doi: 10.1042/bj3380055) (PMID:9931298) (PMCID:PMC1220024)

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 µM and for NADP of 1.9±0.2 µM 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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Arkison, Mr Scott and Barrett, Professor Michael
• Authors: Tetaud, E., Hanau, S., Wekks, J. M., Le Page, R. W.F., Adams, M. J., Arkison, S., and Barrett, M. P.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: Biochemical Journal
• Publisher: Portland Press
• ISSN: 0264-6021
• ISSN (Online): 1470-8728