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Structural determinants of nucleotide coenzyme specificity in the distinctive dinucleotide binding fold of HMG-CoA reductase from Pseudomonas mevalonii.
Biochemistry. 1996 Sep 17; 35(37):11945-50.B

Abstract

The 102-residue small domain of the 428-residue NAD(H)-dependent HMG-CoA reductase of Pseudomonas mevalonii (EC 1.1.1.88) binds NAD(H) at a distinctive, non-Rossmann dinucleotide binding fold. The three-dimensional structure reveals that Asp146 lies close to the 2'-OH of NAD-. To investigate the role of this residue in determination of coenzyme specificity, Asp146 was mutated to Ala, Gly, Ser, and Asn. The mutant enzymes were analyzed for their ability to catalyze the oxidative acylation of mevalonate to HMG-CoA using either the natural coenzyme NAD+ or the alternate coenzyme NADP+. Mutation of Asp146 to Ala or Gly increased the specificity for NADP+, expressed as the ratio of kcat/K(m) for NADP+ to kcat/K(m) for NAD+, 1200-fold (enzyme D146G) and 6700-fold (enzyme D146A). Mutation of Asp146 was accompanied by 565-fold (D146G) and 330-fold (D146A) increases in kcat/K(m) for NADP+ and 2-fold (D146G) and 20-fold (D146A) decreases in kcat/K(m) for NAD+. To further improve NADP+ specificity, Gln147, Leu148, Leu149, or Thr192 of enzyme D146G or D146A was replaced by lysine or arginine, which could stabilize the 2'-phosphate of NADP+. Enzymes D146G/T192K, D146G/T192R, D146G/L148K, D146A/L148K, and D146A/L148R exhibited 3200-, 4500-, 56000-, 72000-, and 83000-fold increases in the specificity for NADP+ relative to the wild-type enzyme.

Authors+Show Affiliations

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

8810898

Citation

Friesen, J A., et al. "Structural Determinants of Nucleotide Coenzyme Specificity in the Distinctive Dinucleotide Binding Fold of HMG-CoA Reductase From Pseudomonas Mevalonii." Biochemistry, vol. 35, no. 37, 1996, pp. 11945-50.
Friesen JA, Lawrence CM, Stauffacher CV, et al. Structural determinants of nucleotide coenzyme specificity in the distinctive dinucleotide binding fold of HMG-CoA reductase from Pseudomonas mevalonii. Biochemistry. 1996;35(37):11945-50.
Friesen, J. A., Lawrence, C. M., Stauffacher, C. V., & Rodwell, V. W. (1996). Structural determinants of nucleotide coenzyme specificity in the distinctive dinucleotide binding fold of HMG-CoA reductase from Pseudomonas mevalonii. Biochemistry, 35(37), 11945-50.
Friesen JA, et al. Structural Determinants of Nucleotide Coenzyme Specificity in the Distinctive Dinucleotide Binding Fold of HMG-CoA Reductase From Pseudomonas Mevalonii. Biochemistry. 1996 Sep 17;35(37):11945-50. PubMed PMID: 8810898.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Structural determinants of nucleotide coenzyme specificity in the distinctive dinucleotide binding fold of HMG-CoA reductase from Pseudomonas mevalonii. AU - Friesen,J A, AU - Lawrence,C M, AU - Stauffacher,C V, AU - Rodwell,V W, PY - 1996/9/17/pubmed PY - 1996/9/17/medline PY - 1996/9/17/entrez SP - 11945 EP - 50 JF - Biochemistry JO - Biochemistry VL - 35 IS - 37 N2 - The 102-residue small domain of the 428-residue NAD(H)-dependent HMG-CoA reductase of Pseudomonas mevalonii (EC 1.1.1.88) binds NAD(H) at a distinctive, non-Rossmann dinucleotide binding fold. The three-dimensional structure reveals that Asp146 lies close to the 2'-OH of NAD-. To investigate the role of this residue in determination of coenzyme specificity, Asp146 was mutated to Ala, Gly, Ser, and Asn. The mutant enzymes were analyzed for their ability to catalyze the oxidative acylation of mevalonate to HMG-CoA using either the natural coenzyme NAD+ or the alternate coenzyme NADP+. Mutation of Asp146 to Ala or Gly increased the specificity for NADP+, expressed as the ratio of kcat/K(m) for NADP+ to kcat/K(m) for NAD+, 1200-fold (enzyme D146G) and 6700-fold (enzyme D146A). Mutation of Asp146 was accompanied by 565-fold (D146G) and 330-fold (D146A) increases in kcat/K(m) for NADP+ and 2-fold (D146G) and 20-fold (D146A) decreases in kcat/K(m) for NAD+. To further improve NADP+ specificity, Gln147, Leu148, Leu149, or Thr192 of enzyme D146G or D146A was replaced by lysine or arginine, which could stabilize the 2'-phosphate of NADP+. Enzymes D146G/T192K, D146G/T192R, D146G/L148K, D146A/L148K, and D146A/L148R exhibited 3200-, 4500-, 56000-, 72000-, and 83000-fold increases in the specificity for NADP+ relative to the wild-type enzyme. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8810898/Structural_determinants_of_nucleotide_coenzyme_specificity_in_the_distinctive_dinucleotide_binding_fold_of_HMG_CoA_reductase_from_Pseudomonas_mevalonii_ L2 - https://doi.org/10.1021/bi9609937 DB - PRIME DP - Unbound Medicine ER -