A comparative structural analysis reveals distinctive features of co-factor binding and substrate specificity in plant aldo-keto reductases

Abstract

Plant aldo-keto reductases of the AKR4C subfamily play key roles during stress and are attractive targets for developing stress-tolerant crops. However, these AKR4Cs show little to no activity with previously-envisioned sugar substrates. We hypothesized a structural basis for the distinctive cofactor binding and substrate specificity of these plant enzymes. To test this, we solved the crystal structure of a novel AKR4C subfamily member, the AKR4C7 from maize, in the apo form and in complex with NADP(+). The binary complex revealed an intermediate state of cofactor binding that preceded closure of Loop B, and also indicated that conformational changes upon substrate binding are required to induce a catalytically-favorable conformation of the active-site pocket. Comparative structural analyses of homologues (AKR1B1, AKR4C8 and AKR4C9) showed that evolutionary redesign of plant AKR4Cs weakened interactions that stabilize the closed conformation of Loop B. This in turn decreased cofactor affinity and altered configuration of the substrate-binding site. We propose that these structural modifications contribute to impairment of sugar reductase activity in favor of other substrates in the plant AKR4C subgroup, and that catalysis involves a three-step process relevant to other AKRs.

Crystal structure of yeast xylose reductase in complex with a novel NADP‐DTT adduct provides insights into substrate recognition and catalysis

“…). For instance, in the plant AKR4 family, specific interactions of a shortened loop 7 that stabilize the closed conformation of the loop were held responsible for the lower cosubstrate affinity and activity of Zm AR in comparison to hAR .…”

Comparison of Glyphosate-Degradation Ability of Aldo-Keto Reductase (AKR4) Proteins in Maize, Soybean and Rice

“…ZmAKR4-expressing E. coli showed the highest glyphosate tolerance, as indicated by the detoxification of most of the glyphosate ( Figure 2 A–C). Previous studies revealed that ZmAKR4 preferred the conversion of sorbitol to glucose in the reaction (sorbitol + NADP + ⇄ glucose + NADPH) [ 31 , 41 ]. Therefore, ZmAKR4 appeared to favour using NADP + to oxidize glyphosate, rather than using NADPH to reduce benzaldehyde.…”