Structural determinant for cold inactivation of rodent L-xylulose reductase.
L-Xylulose reductase (XR) is a homotetramer belonging to the short-chain dehydrogenase/reductase family. Human XR is stable at low temperature, whereas the enzymes of mouse, rat, guinea pig, and hamster are rapidly dissociated into their inactive dimeric forms. In order to identify amino acid residues that cause cold inactivation of the rodent XRs, we have here selected Asp238, Leu242, and Thr244 in the C-terminal regions of rodent XRs and performed site-directed mutagenesis of the residues of mouse XR to the corresponding residues (Glu, Trp, and Cys) of the human enzyme. Cold inactivation was prevented partially by the single mutation of L242W and the double mutation of L242W/T244C, and completely by the double mutation of D238E/L242W. The L242W and L242W/T244C mutants existed in both tetrameric and dimeric forms at low temperature and the D238E/L242W mutant retained its tetrameric structure. No preventive effect was exerted by the mutations of D238E and T244C, which were dissociated into their dimeric forms upon cooling. Crystallographic analysis of human XR revealed that Glu238 and Trp242 contribute to proper orientation of the guanidino group of Arg203 of the same subunit to the C-terminal carboxylate group of Cys244 of another subunit through the neighboring residues, Gln137 and Phe241. Thus, the determinants for cold inactivation of rodent XRs are Asp238 and Leu242 with small side chains, which weaken the salt bridges between Arg203 and the C-terminal carboxylate group, and lead to cold inactivation.
Ishikura
Shuhei
S
Laboratory of Biochemistry, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan.
Usami
Noriyuki
N
El-Kabbani
Ossama
O
Hara
Akira
A
D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003080:Cold Temperature; D018360:Crystallography, X-Ray; D017931:DNA Primers; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D013401:Sugar Alcohol Dehydrogenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification
D017931:DNA Primers; D013401:Sugar Alcohol Dehydrogenases; C023300:L-xylulose reductase