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

Abstract

Aldose reductases (ARs) belonging to the aldo‐keto reductase (AKR) superfamily catalyze the conversion of carbonyl substrates into their respective alcohols. Here we report the crystal structures of the yeast Debaryomyces nepalensis xylose reductase (DnXR, AKR2B10) in the apo form and as a ternary complex with a novel NADP‐DTT adduct. Xylose reductase, a key enzyme in the conversion of xylose to xylitol, has several industrial applications. The enzyme displayed the highest catalytic efficiency for l‐threose (138 ± 7 mm−1·s−1) followed by d‐erythrose (30 ± 3 mm−1·s−1). The crystal structure of the complex reveals a covalent linkage between the C4N atom of the nicotinamide ring of the cosubstrate and the S1 sulfur atom of DTT and provides the first structural evidence for a protein mediated NADP–low‐molecular‐mass thiol adduct. We hypothesize that the formation of the adduct is facilitated by an in‐crystallo Michael addition of the DTT thiolate to the specific conformation of bound NADPH in the active site of DnXR. The interactions between DTT, a four‐carbon sugar alcohol analog, and the enzyme are representative of a near‐cognate product ternary complex and provide significant insights into the structural basis of aldose binding and specificity and the catalytic mechanism of ARs. Database Structural data are available in the PDB under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZCI and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZCM.

Functional and structural properties of pyridoxal reductase (PdxI) from Escherichia coli: a pivotal enzyme in the vitamin B6 salvage pathway

“…Notably, the NADPH hydrogen-donating atom and the carbonyl carbon of PL are separated at a distance of 3.1 Å. Similar distance between NADPH and other carbonyl substrates have been reported in AKRs [33].…”

“…It is worth noting that while a large number of unliganded or binary AKR structures have been solved to date, only a few ternary AKR structures with NADPH or NADP + along with the carbonyl or alcohol have been reported in the literature. Three notable examples are human aldose reductase with bound NADP + and d ‐glyceraldehyde (AKR‐NADP + ‐GAL) (PDB: 3V36) [31], human aldose reductase with bound NADP + and glucose‐6‐phosphate (AKR‐NADP + ‐G6P) (PDB: 2ACQ) [32], and yeast Debaryomyces nepalensis xylose reductase in complex with a NADP and 2,3‐dihydroxy‐1,4‐dithiobutane (PDB: 5ZCM) [33].…”

“…In AKRs, the carbonyl substrate and the NADPH nicotinamide moiety are positioned in close proximity to the four highly conserved catalytic tetrad residues: Asp, Tyr, Lys and His [33][34][35][36][37][38]. In the PdxI structure, Asp, Tyr and Lys residues are strictly conserved (specifically Asp54, Tyr59 and Lys84), while the His residue in AKRs is replaced by Arg126 in PdxI (Figs 8 and 9).…”

Structure–function characterization of an aldo–keto reductase involved in detoxification of the mycotoxin, deoxynivalenol

“…S4 for HPLC chromatograms). To examine the molecular basis for this diastereoselectivity, we superimposed apo-DepB Rleg with the recently solved Debaryomyces nepalensis xylose reductase (PDB ID: 5ZCM) complexed with a DTT-NADP + adduct 52 . In this complex, the geometry of the C4N of NADP + strongly resembles the puckered ring conformation of reduced NADPH.…”

Covalent Adduct Formation as a Strategy for Efficient CO2 Fixation in Crotonyl-CoA Carboxylases/Reductases

“…Covalent adduct formation between the nicotinamide ring of either NADPH or NADH and other substrates has also been reported in other enzymes. − Most of them, however, are competitive inhibitors of the enzyme. Etr’s are also inhibited by an alternative adduct formed between the C4 carbon atom of NAD­(P)H and crotonyl-CoA. , Yet, the function of this C4 adduct is clearly different from the C2 adduct because its formation diminishes the catalytic efficiency of NAD­(P)H dependent enzymes.…”