Biochemical characterization of an L-Xylulose reductase from Neurospora crassa

Abstract

An l-xylulose reductase identified from the genome sequence of the filamentous fungus Neurospora crassa was heterologously expressed in Escherichia coli as a His(6) tag fusion protein, purified, and characterized. The enzyme may be used in the production of xylitol from the major pentose components of hemicellulosic waste, d-xylose and l-arabinose.

FIG. 1.

SDS-PAGE analysis of purified, heterologously expressed N. crassa LXR. Lanes: 1 and 8, MW standard; 2, purified LXR after His₆ tag cleavage; 3, purified His₆-LXR fusion; 4 and 5, soluble and insoluble fractions, respectively, of cell lysate expressing His₆-LXR; 6 and 7, soluble and insoluble fractions, respectively, of cell lysate harboring empty plasmid (6). MW, molecular weight.

FIG. 2.

(A) The temperature optimum for the reductase activity of N. crassa LXR was determined to be ∼37°C when assayed between temperatures from 15 to 60°C. (B) Thermal inactivation of N. crassa LXR at 45°C. The heat inactivation followed first-order kinetics with a half-life of 12.9 min. (C) The pH optimum for the reductase activity (▪) of N. crassa LXR was at 7.0 and at 9.0 for oxidase activity (▵). The enzyme shows a broad range for activity with >50% reductase activity between pH 4.5 and 8.5.

FIG. 3.

Comparison of the crystal structure of human LXR (A) and homology model for N. crassa LXR with NADP (B). The catalytic triads (Ser, Tyr, and Lys) are shown and are approximately similar in position and orientation. Position of NADP differs in the two possibly because of Tyr57 in N. crassa LXR, offering steric hindrance to the adenine group of the cofactor. The longer N terminal is distant from the catalytic site, possibly accounting for minimal effect of the His₆ tag.