A Novel NADH-linked L-Xylulose Reductase in the L-Arabinose Catabolic Pathway of Yeast

Abstract

An NADH-dependent L-xylulose reductase and the corresponding gene were identified from the yeast Ambrosiozyma monospora. The enzyme is part of the yeast pathway for L-arabinose catabolism. A fungal pathway for L-arabinose utilization has been described previously for molds. In this pathway L-arabinose is sequentially converted to L-arabinitol, L-xylulose, xylitol, and D-xylulose and enters the pentose phosphate pathway as D-xylulose 5-phosphate. In molds the reductions are NADPH-linked, and the oxidations are NAD ؉ -linked. Here we show that in A. monospora the pathway is similar, i.e. it has the same two reduction and two oxidation reactions, but the reduction by L-xylulose reductase is not performed by a strictly NADPH-dependent enzyme as in molds but by a strictly NADH-dependent enzyme. The ALX1 gene encoding the NADH-dependent Lxylulose reductase is strongly expressed during growth on L-arabinose as shown by Northern analysis. The gene was functionally overexpressed in Saccharomyces cerevisiae and the purified His-tagged protein characterized. The reversible enzyme converts Lxylulose to xylitol. It also converts D-ribulose to Darabinitol but has no activity with L-arabinitol or adonitol, i.e. it is specific for sugar alcohols where, in a Fischer projection, the hydroxyl group of the C-2 is in the L-configuration and the hydroxyl group of C-3 is in the D-configuration. It also has no activity with C-6 sugars or sugar alcohols. The K m values for L-xylulose and D-ribulose are 9.6 and 4.7 mM, respectively. To our knowledge this is the first report of an NADH-linked L-xylulose reductase.

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

“…This pathway consists of two NAD + -linked oxidations and two NADPH-linked reductions, resulting in a redox cofactor imbalance under anaerobic conditions (Dien et al 1996). In contrast to the strictly NADP(H)-dependent L-xylulose reductase described for the molds P. chrysogenum (Chiang and Knight 1960), A. niger (Witteveen et al 1994), and Trichoderma reesei (Richard et al 2002), the L-xylulose reductase of the yeast Ambrosiozyma monospora has been reported to be strictly NADH-dependent (Verho et al 2004). It is unknown whether this is a general characteristic of this enzyme in L-arabinose-metabolizing yeasts.…”

“…Another way that has been proposed to ameliorate problems with cofactor imbalances is expression of the aldose reductase gene from Pi. stipitis (Rizzi et al 1988) that also accepts NADH as cofactor, or the gene encoding NADH-dependent L-xylulose reductase (ALX1) from Ambrosiozyma monospora (Verho et al 2004). …”

Cloning and Overexpression of the Xylitol Dehydrogenase Gene from Bacillus pallidus and Its Application to L-Xylulose Production

“…[1][2][3][4][5][6][7] Erwinia uredovora (later renamed Pantoea ananatis) is the only bacterium known to have an activity related to L-xylulose formation from xylitol. Many studies have been conducted on the mechanisms and properties of the enzyme in this organism.…”

“…This result showed the size to be comparable with related enzymes from other sources, which appeare to have approximate sizes of 26-40 kDa on SDS-PAGE analysis. 2,3,6,11,16) We also constructed C-terminal His-tagged XDH to facilitate purification, but XDH activity was significantly lower (data not shown). Hence the non-Histagged enzyme was used throughout this study.…”

A Novel α-Ketoglutaric Semialdehyde Dehydrogenase

“…The D-xylulose 5-phosphate is then further metabolized in the pentose-phosphate pathway. In a recently characterized fungal pathway (3)(4)(5), L-arabinose is also converted to D-xylulose 5-phosphate but through different intermediates by two reductases, two dehydrogenases, and a kinase. In the hypothetical pathway(s) involving nonphosphorylated intermediates in bacteria ( Fig.…”