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6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme.
Biochem J. 1999 Feb 15; 338 (Pt 1):55-60.BJ

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.

Authors+Show Affiliations

Laboratoire de Biologie Moléculaire et Immunologie de Parasites Protozoaires, UPRESA-5016 CNRS, Université Bordeaux II, France.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9931298

Citation

Tetaud, E, et al. "6-Phosphogluconate Dehydrogenase From Lactococcus Lactis: a Role for Arginine Residues in Binding Substrate and Coenzyme." The Biochemical Journal, vol. 338 (Pt 1), 1999, pp. 55-60.
Tetaud E, Hanau S, Wells JM, et al. 6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme. Biochem J. 1999;338 (Pt 1):55-60.
Tetaud, E., Hanau, S., Wells, J. M., Le Page, R. W., Adams, M. J., Arkison, S., & Barrett, M. P. (1999). 6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme. The Biochemical Journal, 338 (Pt 1), 55-60.
Tetaud E, et al. 6-Phosphogluconate Dehydrogenase From Lactococcus Lactis: a Role for Arginine Residues in Binding Substrate and Coenzyme. Biochem J. 1999 Feb 15;338 (Pt 1):55-60. PubMed PMID: 9931298.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme. AU - Tetaud,E, AU - Hanau,S, AU - Wells,J M, AU - Le Page,R W, AU - Adams,M J, AU - Arkison,S, AU - Barrett,M P, PY - 1999/2/5/pubmed PY - 1999/2/5/medline PY - 1999/2/5/entrez SP - 55 EP - 60 JF - The Biochemical journal JO - Biochem. J. VL - 338 (Pt 1) N2 - 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. SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/9931298/6_Phosphogluconate_dehydrogenase_from_Lactococcus_lactis:_a_role_for_arginine_residues_in_binding_substrate_and_coenzyme_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/9931298/ DB - PRIME DP - Unbound Medicine ER -