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

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 5ZCI and 5ZCM.

A temperature-responsive gene in sorghum encodes a glycine-rich protein that interacts with calmodulin.

Imposition of different biotic and abiotic stress conditions results in an increase in intracellular levels of Ca2+ which is sensed by various sensor proteins. Calmodulin (CaM) is one of the best studied transducers of Ca2+ signals. CaM undergoes conformational changes upon binding to Ca2+ and interacts with different types of proteins, thereby, regulating their activities. The present study reports the cloning and characterization of a sorghum cDNA encoding a protein (SbGRBP) that shows homology to glycine-rich RNA-binding proteins. The expression of SbGRBP in the sorghum seedlings is modulated by heat stress. The SbGRBP protein is localized in the nucleus as well as in cytosol, and shows interaction with CaM that requires the presence of Ca2+. SbGRBP depicts binding to single- and also double-stranded DNA. Fluorescence spectroscopic analyses suggest that interaction of SbGRBP with nucleic acids may be modulated after binding with CaM. To our knowledge, this is the first study to provide evidence for interaction of a stress regulated glycine-rich RNA-binding protein with CaM.