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Altering dimer contacts in xylose reductase from Candida tenuis by site-directed mutagenesis: structural and functional properties of R180A mutant.
Chem Biol Interact. 2003 Feb 01; 143-144:523-32.CB

Abstract

Xylose reductase (XR) from Candida tenuis (CtXR) is a structurally characterized member of family 2 of the aldo-keto reductase (AKR) superfamily of proteins, its family designation being AKR2B5. The enzyme is composed of two identical subunits that contact each other in a largely hydrated, predominantly polar interface. An important question not clearly answered by CtXR structure pertains to the relationship of oligomerization and enzyme activity. In an effort to destabilize the wild-type dimer, the most important secondary structural element of the CtXR interface, the alpha5 helix, was altered by site-directed mutagenesis. Ala-173 and Leu-174 were replaced individually by arginine, and Arg-180 was changed into alanine. A173R and L174R mutants did not fold properly during recombinant protein production in Escherichia coli and could not be isolated. Like the wild type, the R180A mutant is a dimer in solution which does not dissociate into subunits under mild urea conditions (</=2 M). Catalytic efficiency (k(cat)/K(xylose)) and turnover number (k(cat)) of the R180A mutant for NADH-dependent reduction of D-xylose are both approximately 2.5-fold decreased compared to corresponding kinetic parameters of the wild type. Differences in kinetic isotope effects for the mutant (Dk(cat)=1.0; Dk(cat)/K(xylose)=1.9) and the wild type (Dk(cat)=1.5; Dk(cat)/K(xylose)=2.8) suggest subtle changes in catalytic function as result of the mutation. Therefore, altering interactions at the dimer interface may have long range effects that were not predictable from the X-ray structure.

Authors+Show Affiliations

Institute of Biotechnology, Graz University of Technology, Petersgasse 12/I, A-8010, Graz, Austria.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

12604238

Citation

Klimacek, Mario, et al. "Altering Dimer Contacts in Xylose Reductase From Candida Tenuis By Site-directed Mutagenesis: Structural and Functional Properties of R180A Mutant." Chemico-biological Interactions, vol. 143-144, 2003, pp. 523-32.
Klimacek M, Wührer F, Kavanagh KL, et al. Altering dimer contacts in xylose reductase from Candida tenuis by site-directed mutagenesis: structural and functional properties of R180A mutant. Chem Biol Interact. 2003;143-144:523-32.
Klimacek, M., Wührer, F., Kavanagh, K. L., Wilson, D. K., & Nidetzky, B. (2003). Altering dimer contacts in xylose reductase from Candida tenuis by site-directed mutagenesis: structural and functional properties of R180A mutant. Chemico-biological Interactions, 143-144, 523-32.
Klimacek M, et al. Altering Dimer Contacts in Xylose Reductase From Candida Tenuis By Site-directed Mutagenesis: Structural and Functional Properties of R180A Mutant. Chem Biol Interact. 2003 Feb 1;143-144:523-32. PubMed PMID: 12604238.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Altering dimer contacts in xylose reductase from Candida tenuis by site-directed mutagenesis: structural and functional properties of R180A mutant. AU - Klimacek,Mario, AU - Wührer,Franz, AU - Kavanagh,Kathryn L, AU - Wilson,David K, AU - Nidetzky,Bernd, PY - 2003/2/27/pubmed PY - 2003/5/13/medline PY - 2003/2/27/entrez SP - 523 EP - 32 JF - Chemico-biological interactions JO - Chem. Biol. Interact. VL - 143-144 N2 - Xylose reductase (XR) from Candida tenuis (CtXR) is a structurally characterized member of family 2 of the aldo-keto reductase (AKR) superfamily of proteins, its family designation being AKR2B5. The enzyme is composed of two identical subunits that contact each other in a largely hydrated, predominantly polar interface. An important question not clearly answered by CtXR structure pertains to the relationship of oligomerization and enzyme activity. In an effort to destabilize the wild-type dimer, the most important secondary structural element of the CtXR interface, the alpha5 helix, was altered by site-directed mutagenesis. Ala-173 and Leu-174 were replaced individually by arginine, and Arg-180 was changed into alanine. A173R and L174R mutants did not fold properly during recombinant protein production in Escherichia coli and could not be isolated. Like the wild type, the R180A mutant is a dimer in solution which does not dissociate into subunits under mild urea conditions (</=2 M). Catalytic efficiency (k(cat)/K(xylose)) and turnover number (k(cat)) of the R180A mutant for NADH-dependent reduction of D-xylose are both approximately 2.5-fold decreased compared to corresponding kinetic parameters of the wild type. Differences in kinetic isotope effects for the mutant (Dk(cat)=1.0; Dk(cat)/K(xylose)=1.9) and the wild type (Dk(cat)=1.5; Dk(cat)/K(xylose)=2.8) suggest subtle changes in catalytic function as result of the mutation. Therefore, altering interactions at the dimer interface may have long range effects that were not predictable from the X-ray structure. SN - 0009-2797 UR - https://www.unboundmedicine.com/medline/citation/12604238/Altering_dimer_contacts_in_xylose_reductase_from_Candida_tenuis_by_site_directed_mutagenesis:_structural_and_functional_properties_of_R180A_mutant_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0009279702002132 DB - PRIME DP - Unbound Medicine ER -