Multiple Forms of Xylose Reductase in Candida intermedia:  Comparison of Their Functional Properties Using Quantitative Structure−Activity Relationships, Steady-State Kinetic Analysis, and pH Studies

Abstract

The xylose-fermenting yeast Candida intermedia produces two isoforms of xylose reductase: one is NADPH-dependent (monospecific xylose reductase; msXR), and another is shown here to prefer NADH approximately 4-fold over NADPH (dual specific xylose reductase; dsXR). To compare the functional properties of the isozymes, a steady-state kinetic analysis for the reaction d-xylose + NAD(P)H + H(+) <--> xylitol + NAD(P)(+) was carried out and specificity constants (k(cat)/K(aldehyde)) were measured for the reduction of a series of aldehydes differing in side-chain size as well as hydrogen-bonding capabilities with the substrate binding pocket of the enzyme. dsXR binds NAD(P)(+) (K(iNAD+) = 70 microM; K(iNADP+) = 55 microM) weakly and NADH (K(i) = 8 microM) about as tightly as NADPH (K(i) = 14 microM). msXR shows uniform binding of NADPH and NADP(+) (K(iNADP+) approximately K(iNADPH) = 20 microM). A quantitative structure-activity relationship analysis was carried out by correlating logarithmic k(cat)/K(aldehyde) values for dsXR with corresponding logarithmic k(cat)/K(aldehyde) values for msXR. This correlation is linear with a slope of approximately 1 (r (2) = 0.912), indicating that no isozyme-related pattern of substrate specificity prevails and aldehyde-binding modes are identical in both XR forms. Binary complexes of dsXR-NADH and msXR-NADPH show the same macroscopic pK of approximately 9.0-9.5, above which the activity is lost in both enzymes. A lower pK of 7.4 is seen for dsXR-NADPH. Specificity for NADH and greater binding affinity for NAD(P)H than NAD(P)(+) are thus the main features of enzymic function that distinguish dsXR from msXR.

Heterologous Expression, Purification, and Characterization of a Highly Active Xylose Reductase from Neurospora crassa

“…Xylose reductase (XR) is commonly found in yeasts and filamentous fungi, often with several isozymes in one species (21,24,33). XR catalyzes the first step of D-xylose metabolism, reducing xylose to xylitol with concomitant NAD(P)H oxidation.…”

“…In general, XR is specific for NADPH, but in some cases, it utilizes both NADPH and NADH (7,21,23,30) and in at least one case prefers NADH over NADPH (20). The difference in cofactor specificities is proposed to maintain the redox balance between nicotinamide cofactors under a variety of growth conditions (8,24). Based on sequence and structure similarities, XR belongs to the aldose reductase family (EC 1.1.1.21).…”

“…The genetic sequences of many XRs have been determined and several have been cloned and expressed in a variety of hosts (1,7,9,13,18,20,21,23,24,33), but many XR genes are still uncharacterized or unidentified. Such is the case with Neurospora crassa, which is known to convert plant biomass to ethanol (3,22,27), suggesting the presence of D-xylose-metabolizing enzymes (28) though no XR gene has been identified.…”

“…This difference is a function of N. crassa XR having both a higher k cat (3,600 min Ϫ1 , compared to 312 min Ϫ1 ) and a lower K m (1.8 M, compared to 16 M) with NADPH than with NADH. (18,20,23,24). Table 3 displays the kinetic characteristics of purified and characterized XRs from Candida intermedia, Candida parapsilosis, C. tropicalis, C. tenuis, Pachysolen tannophilus, Pichia stipitus, and Saccharomyces cerevisiae (4,7,11,20,21,23,24,30,33).…”

Molecular cloning and functional expression of a novelNeurospora crassa xylose reductase inSaccharomyces cerevisiae in the development of a xylose fermenting strain

“…Filamentous fungi have been shown to possess several isozymes in one species (Yokoyama et al, 1995b;Mayr et al, 2000;Nidetzky et al, 2003). Knowledge of as many isozymes as possible would help in understanding their function and could lead to novel characterisations.…”