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Applied and Environmental Microbiology, October 2005, p. 6390-6393, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6390-6393.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Engineering Candida tenuis Xylose Reductase for Improved Utilization of NADH: Antagonistic Effects of Multiple Side Chain Replacements and Performance of Site-Directed Mutants under Simulated In Vivo Conditions

Barbara Petschacher and Bernd Nidetzky^*

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria

Received 9 March 2005/ Accepted 28 April 2005

Top Abstract Introduction References

 
Six single- and multiple-site variants of Candida tenuis xylose reductase that were engineered to have side chain replacements in the coenzyme 2'-phosphate binding pocket were tested for NADPH versus NADH selectivity (R_sel) in the presence of physiological reactant concentrations. The experimental R_sel values agreed well with predictions from a kinetic mechanism describing mixed alternative coenzyme utilization. The Lys-274Arg and Arg-280His substitutions, which individually improved wild-type R_sel 50- and 20-fold, respectively, had opposing structural effects when they were combined in a double mutant.

Top Abstract Introduction References

 
Many efforts have been made to develop an industrial process for the conversion of xylose into fuel ethanol using microbial fermentation (5, 6, 8). Saccharomyces cerevisiae is a prime candidate for alcohol production but cannot assimilate xylose in its wild-type form. Expansion of the substrate range of S. cerevisiae to include xylose was achieved by introducing the following missing steps of the functional xylose metabolism in other yeasts: reduction of xylose to xylitol by xylose reductase (XR) and oxidation of xylitol to xylulose by xylitol dehydrogenase (XDH). However, engineered S. cerevisiae strains expressing heterologous genes of the two oxidoreductases displayed high levels of xylitol excretion and low ethanol yields (6). This shortcoming was due to the impairment of the redox balance resulting from the different coenzyme specificities of XR, which is mainly NADPH dependent, and XDH, which is specific for NAD⁺ (3). Different parts of the S. cerevisiae central metabolism may be engineered to enable partial regeneration of the NADPH (18) and NAD⁺ (15, 16) consumed for xylose conversion, but ideally the imbalance is avoided at its source. Kuyper et al. (9-11) have shown the large impact of replacing the oxidoreductive two-step isomerization of xylose with a direct isomerase-catalyzed transformation that is intrinsically redox neutral because it does not require coenzyme. Staying with the two-step metabolism, coenzyme recycling during xylose assimilation could be achieved by making the XR NADH dependent (14) or converting XDH into an NADP⁺-specific enzyme (13, 19). We used structure-guided site-directed mutagenesis of Candida tenuis XR to change the coenzyme selectivity of this enzyme from a 34-fold preference for NADPH in the wild type to a 6-fold preference for NADH in a Lys-274Arg Asn-276Asp double mutant (Fig. 1). Protein engineering normally generates many more enzyme variants than can possibly be assessed in fermentation studies. This emphasizes the importance of rigorous evaluation of mutants in vitro under physiologically relevant conditions. We describe here a combined experimental and theoretical approach which is extremely useful for characterizing precisely and quickly the coenzyme selectivity of C. tenuis XR and selected mutants of this enzyme under mixed NADPH and NADH utilization conditions. We also combined previously reported C. tenuis XR single-site mutants into novel double and triple mutants and found that multiple side chain replacements are distinguished by their additive effects (Lys-274Arg Asn-276Asp) and antagonistic effects (Lys-274Arg Arg-280His) on coenzyme selectivity.

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FIG. 1. Coenzyme binding to wild-type C. tenuis XR. The interactions with adenosine ribose moieties in NADP+ (left panel) and NAD+ (right panel) are shown.

Site-directed mutagenesis was performed by using previously described protocols (7, 14). The mutated genes encoding Lys-274Arg Arg-280His (K274R R280H) and Lys-274Arg Asn-276Asp Agr-280His (K274R N276D R280H) were generated by inverse PCR using oligonucleotide primer 5'-CCAGAGCATCTAGTCCAAAACAGAAGTTTC-3' for the forward direction. In the reverse direction, the following primers were used: 5'-GAGGTTAGACCTTGGAATGACAGC-3' (K274R R280H) and 5'-GAGGTCAGACCTTGGAATGACAGC-3' (K274R N276D R280H)

Mutant C. tenuis XRs were produced in Escherichia coli and purified to apparent homogeneity using methods described elsewhere in detail (12, 14). Steady-state kinetic analysis of the initial rates of NADPH- or NADH-dependent reduction of xylose was performed as reported recently (14). The kinetic parameters of the K274R R280H and K274R N276D R280H mutants are summarized in Table 1 along with the wild-type data. The differences in kinetic parameters for the wild type and the mutants are best ascribed to their combined effects on the coenzyme selectivity (R_sel).

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TABLE 1. Kinetic parameters for NAD(P)H-dependent reduction of xylose by C. tenuis XR and two mutants of this enzymea

The parameter R_sel is defined as the ratio of catalytic efficiencies, k_cat/(K_ia K_b), for reactions with NADPH and NADH, where k_cat is the turnover number, K_ia is the apparent binding constant for the coenzyme, and K_b is the Michaelis constant for xylose. In Table 1, K_a is the Michaelis constant for the coenzyme. The double and triple mutants showed about the same R_sel value, 3. The corresponding R_sel values for the K274R, R280H, and K274R N276D mutants are 0.7, 4.2, and 0.2, respectively (14). Interestingly, therefore, incorporation of the side chain replacement Arg-280His into K274R and K274R N276D mutants resulted in 4.3-fold and 15-fold increases in R_sel, indicating that the quantitative effect of the additional mutation on the mutated enzymes was strongly antagonistic. The results suggest that there are opposing structural effects of the Lys-274Arg mutation, alone or in combination with Asn-276Asp, and the Arg-280His mutation. The crystal structures of the wild type bound to NADP⁺ and NAD⁺ (Fig. 1) provide a possible explanation: the effect of steric hindrance on the 2'-phosphate group of NADP⁺, generated by the introduction of the side chain of Arg-274, could be strongly reduced when the side chain of Arg-280 is replaced by the smaller side chain of a histidine. It seems possible, therefore, that R_sel for the K274R N276D double mutant presents a limiting case so that it may be difficult to further improve the selectivity of C. tenuis XR by structure-guided engineering of the coenzyme binding site.

The catalytic efficiencies shown in Table 1 reflect extrapolation to conditions in which the concentrations of coenzyme and substrate are both limiting. They do not clearly reflect the situation in a living yeast cell, in which the intracellular concentrations of NADPH (508 µM) and NADH (185 µM) are almost saturating in the steady state (1, 17). The scenario of mixed coenzyme utilization is also not accounted for by a kinetic mechanism for the enzymatic reaction with a single coenzyme. We therefore developed an experimental test of coenzyme selectivity under conditions in which both coenzymes are added at the same time at their purported in vivo concentrations. The level of xylose used was 20 g/liter (113 mM), which is within the range of reported physiological concentrations of this sugar (4). Figure 2 explains the principle of the assay and shows typical results, which in all cases are means of triplicate experiments. The reaction was started by addition of enzyme (1.6 U/ml) and was allowed to proceed until about one-half the total coenzyme concentration was depleted. Note the linear decrease in absorbance over time for the duration of the assay. After inactivation of the enzyme by heating to 99°C for 30 s, 100 mM sodium formate and 9 U/ml Candida boidinii formate dehydrogenase were added to specifically reduce the NAD⁺ produced by the action of XR. Appropriate controls were included, and dilution due to reactant additions was taken into account.

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FIG. 2. Experimental assay for determination of the coenzyme selectivities of C. tenuis XR and mutants of this enzyme. Values for the K274R N276D double mutant are shown. The arrows indicate the time of addition of xylose reductase (arrow a), the time that the reaction was stopped by heating (arrow b), the time of addition of formate (arrow c), and the time of addition of C. bodidinii formate dehydrogenase (arrow d).

The coenzyme selectivity (R'_sel) of each mutant was calculated using the experimental data and equation 1, and the results are summarized in Fig. 3.

(1)

The K274R N276D mutant gave the best value for R'_sel (1.5); thus, it utilized NADH about as efficiently as it utilized NADPH. It is important to notice that the observed pattern of coenzyme selectivities across the series of C. tenuis XR mutants was not identical to the pattern derived from a comparison of kinetic R_sel values. The K274R mutant is an interesting example because its R'_sel value (9.2) and R_sel value (0.7) differed almost 13-fold, stressing the value of the experimental analysis shown in Fig. 2.

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FIG. 3. Experimental selectivities can be correctly predicted from a kinetic model for mixed alternative coenzyme utilization. The experimental R'sel values were obtained using equation 1. The standard deviations of R'sel are 20% for the wild type, 9% for the K274R mutant, and <5% for all other mutants. R'sel values were calculated using equation 2.

In an effort to provide a better correlation between the kinetic parameters for the enzymatic reaction and the experimental value of R'_sel, we used equation 2. This equation describes mixed alternative coenzyme utilization during xylose reduction by C. tenuis XR and mutants of this enzyme. It assumes an ordered two-substrate reaction mechanism of the enzyme in which coenzyme binds first (14). It was adapted from the work of Banta et al. (2) with the justified simplification that xylose does not show substrate inhibition at the concentrations used. In equation 2, v_xylitol is the initial rate of xylitol production; the superscripts x and y indicate NADH and NADPH, respectively; and E_t is the total molarity of the enzyme (4 µM). The value of R'_sel (v_NADPH/v_NADH, where v is an initial rate) can be calculated with equation 2 using the known kinetic parameters for NADPH- and NADH-dependent reactions, as shown in Table 1 and reference 14, and the reactant concentrations employed in Fig. 2. The results are summarized in Fig. 3, in which they are plotted along with the experimental values.

(2)

where

A linear fit of the data in Fig. 3 gave a slope of 1.37 with a good correlation coefficient, 0.944. In summary, the results show that physiological coenzyme selectivities of dual-specific NADH- and NADPH-dependent reductases can be relatively accurately determined experimentally using a simple in vitro assay. The R'_sel values derived from an actual conversion experiment are completely consistent with predictions from an expanded kinetic mechanism. They provide a useful and sound basis for selecting suitable C. tenuis XR mutants for future xylose fermentation studies using engineered S. cerevisiae.

 
The Austrian Science Funds (FWF project 15208 to B.N.) provided financial support.

 
* Corresponding author. Mailing address: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria. Phone: 43-316-873-8400. Fax: 43-316-873-8434. E-mail: bernd.nidetzky{at}tugraz.at.

Top Abstract Introduction References

 

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Applied and Environmental Microbiology, October 2005, p. 6390-6393, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6390-6393.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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