Characterization of d-xylose reductase, XyrB, from Aspergillus niger

Abstract

d-xylose reductase is a member of the aldo-keto reductase family, and is involved in d-xylose and l-arabinose conversion through the Pentose Catabolic Pathway (PCP) in fungi. In this study, we biochemically characterized a newly identified second d-xylose reductase (XyrB) from Aspergillus niger. This NADPH-dependent reductase is able to efficiently convert d-xylose and l-arabinose, and it has the highest affinity for these sugars of all currently known fungal pentose reductases. A combination of biochemical data, transcriptomics and phylogenetic analysis further illustrated the role of XyrB in the PCP. Enzymes: D-xylose reductase (EC 1.1.1.307), L-arabinose reductase (EC 1.1.1.21).

Keywords: AKR, aldo-keto reductase; Aspergillus niger; CtXR, Candida tenuis xylose reductase; GCY1/YPR1, yeast glycerol dehydrogenases; GldB, filamentous fungal glycerol dehydrogenase; IPTG, isopropyl β-D-1-thiogalactopyranoside; LB, Luria Bertani; LadA, l-arabitol dehydrogenase; LarA, l-arabinose reductase; LxrA, LxrB, l-xylulose reductase; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced; PBS, phosphate buffered saline; PCP, Pentose Catabolic Pathway; PCR, polymerase chain reaction; PPP, Pentose Phosphate Pathway; PRD1, pentose reductase; Pentose Catabolic Pathway; SdhA, sorbitol dehydrogenase; XdhA, xylitol dehydrogenase; XkiA, d-xylulose kinase; XyrA, XyrB, d-xylose reductase; d-xylose reductase.

Fig. 1

Pentose Catabolic Pathway in Aspergillus niger. Enzymes involved in the pathway: LarA= l-arabinose reductase, LadA = l-arabitol dehydrogenase, LxrA, LxrB = l-xylulose reductase SdhA = sorbitol dehydrogenase, XyrA, XyrB = d-xylose reductase (the main topic of this study), XdhA = xylitol dehydrogenase, XkiA = d-xylulose kinase; PPP = Pentose Phosphate Pathway. Gene IDs are indicated in brackets. Based on Ref. [4].

Fig. 2

Phylogenetic analysis of pentose reductases and related genes. The tree is a representative Maximum Likelihood tree. Bootstrap values (50 or larger) are indicated on the nodes that are supported by the Maximum Likelihood, Neighbor Joining and Minimal Evolution algorithms in this order. XyrA/XyrB/XYR1 = d-xylose reductase, LarA = l-arabinose reductase, PRD1 = pentose reductase, GCY1/YPR1 = yeast glycerol dehydrogenases, GldB = filamentous fungal glycerol dehydrogenase. Colors reflect the taxonomic group of the fungi. Blue = Eurotiomycetes, red = Sordariomycetes, green = Leotiomycetes, orange = Dothidiomycetes, pink = Saccharomycetes. Amino acid sequences used as a query in a BlastP search are in larger font and boldface.

Fig. 3

Expression profiles for d-xylose reductase xyrA, d-xylose reductase xyrB and l-arabinose reductase larA from A. niger. Expression values of the genes (based on normalized probe intensities) were obtained from previously published transcriptome (microarray) study [8]. If only a carbon source is mentioned, than this is the expression value of a wild type strain transferred for 2 h to that carbon source. Glc = d-glucose, frc = d-fructose, gal = d-galactose, man = d-mannose, xyl = d-xylose, ara = d-arabinose, rha = l-rhamnose, galA = d-galacturonic acid. ΔxlnR = xlnR deletion strain transferred for 2 h to d-xylose, ΔaraR = araR deletion transferred for 2 h to l-arabinose. The error bars represent standard deviation of biological duplicates.

Fig. 4

Kinetic characterization of XyrB with different substrates: d-xylose, l-arabinose and d-galactose. The Michaelis-Menten constants were estimated from fitting the Michaelis-Menten kinetics to the data points. Vertical bars represent standard deviation from three assay repeats.