Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases

Abstract

Xylose reductase (XR; AKR2B5) is an unusual member of aldoketo reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD + to 1.80 Å resolution (where 1 Å = 0.1 nm) with a crystallographic R-factor of 18.3 %. A comparison of the NAD + -and the previously determined NADP + -bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2 -phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn 276 , which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu 227 on a short rigid helix, which makes hydrogen bonds to both the 2 -and 3 -hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3 -endo conformation rather than the 2 -endo conformation seen in the NADP + -bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.

Computational design of Candida boidinii xylose reductase for altered cofactor specificity

“…27,53,72 We defined Positions 1-3 as the residues that are aligned to K274, S275, and N276, respectively. Positions 1-3 are nearby the phosphate group in NADPH, but are over 12Å from the hydride transfer site in the catalytic mechanism, highlighting that these positions affect cofactor specificity and affinity but are not directly involved in the reaction.…”

“…The increased hydrophobicity of the methionine side chain relative to lysine may imply that the orientation of the methionine side chain with respect to bulk water is not favored. 27,53 In Figure 1(B), no hydrogen bonding interactions were present between NADH and the design positions chosen in CbXR. In contrast, as shown in Figure 4, the best computationally-derived design, CbXR-EDS (involving three point mutations K272E, S273D, and N274S) improved the interaction energy by À181 kcal/ mol while forming a number of new hydrogen bonds between CbXR and the NADH.…”

“…53,54 The AKR superfamily shares a common (a/ ß) 8 -barrel fold without a Rossmann-fold motif and their members show varied preferences for NADPH over NADH. 55 The active site, conserved in both structure and sequence in nearly all AKRs, is situated in a deep cavity inside the (a/ß) 8 barrel, and is defined by a tetrad of catalytic residues.…”

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+

“…The K274R mutant bound to NADP + or NAD + folds into the (b/a) 8 barrel previously described for wild-type CtXR [4,5]. There were four K274R molecules, two enzyme dimers, within the respective crystallographical asymmetric units.…”

“…Family 2 is unusual among the 14 AKR families currently recognized because it includes dual specific NADPH/NADH-dependent reductases as well as NADPH-specific enzymes. Crystal structures of wild-type CtXR bound to NADP + and NAD + show that a conformational change of the side chain of Glu-227, to make strong interactions with the 2 0 -OH and 3 0 -OH groups when the cofactor 2 0 -phosphate group is lacking, is most critical for utilization of NADH [4,5]. Glu-227 of CtXR is widely conserved among the dual specific AKRs of family 2.…”