Fine tuning of coenzyme specificity in family 2 aldo‐keto reductases revealed by crystal structures of the Lys‐274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD⁺ and NADP⁺

Abstract

Aldo-keto reductases of family 2 employ single site replacement Lys fi Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274 fi Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD + and NADP + were determined at a resolution of 2.4 and 2.3 Å , respectively. Due to steric conflicts in the NADP + -bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2 0 -phosphate and 3 0 -hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P) + in the wild-type remains partly disordered in the NADP + -bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.

Computational design of Candida boidinii xylose reductase for altered cofactor specificity

“…The wild-type lysine in this position provides a positive charge for NADPH binding and the mutation of this residue to Arg was previously shown to change the cofactor specificity of CtXR from NADPH to NADH. 26,27 As negative controls, we also constructed three mutants not predicted by IPRO (CbXR-RNI, -KKG, -RHC).…”

“…Cofactor specificity of this enzyme is markedly influenced by different amino acid substitutions in three design positions. Replacement of Lys-272 by Arg, which was previously shown to completely reverse nicotinamide cofactor specificity in CtXR, 26 also yielded NADH activity in CbXR while weakening the NADPH-linked catalytic activity (by approximately fivefold; see Table IV). While this mutant did not make the top 10 predicted designs, offline interaction energy calculations showed a significant À60 kcal/mol (26%) improvement in interaction energy toward NADH relative to the wild-type CbXR.…”

“…Alignment of AKRs reveals a conserved Lys residue near position 274 (amino acid position 274 in CtXR; position 272 in CbXR), which plays a critical role in cofactor binding. 26 One notable exception is the presence of an Arg residue rather than Lys at position 276 of the XR from C. parapsilosis, which prefers NADH as its cofactor. 60 Leitgeb et al showed that replacement of Lys-274 with Arg in CtXR results in reversal of cofactor specificity for NADH over NADPH.…”

“…Site-directed mutagenesisbased studies also successfully pinpointed sets of mutations leading to complete reversal of Candida tenuis xylose reductase (CtXR) cofactor specificity from NADPH to NADH (reduced form of NAD þ ). 26,27 Similarly, Liang et al 28 used a semirational approach called combinatorial active site saturation (CASTing) to switch cofactor preference from NADPH to NADH in PsXR.…”

“…60 Leitgeb et al showed that replacement of Lys-274 with Arg in CtXR results in reversal of cofactor specificity for NADH over NADPH. 26 Xylitol has been listed among the top value-added platform chemical products of biomass refining. 61 The production of xylitol from xylose by engineered Escherichia coli growing on glucose and expressing a xylose reductase from either C. boidinii, C. tenuis, P. stipitis, or Saccharomyces cerevisiae was recently studied.…”