Reversal of coenzyme specificity and improvement of catalytic efficiency of <Emphasis Type="Italic">Pichia</Emphasis><Emphasis Type="Italic">stipitis</Emphasis> xylose reductase by rational site-directed mutagenesis

A major problem when xylose is used for ethanol production is the intercellular redox imbalance arising from different coenzyme specificities of xylose reductase (XR) and xylitol dehydrogenase. The residue Lys21 in XR from Pichia stipitis was subjected to site-directed mutagenesis to alter its coenzyme specificity. The N272D mutant exhibited improved catalytic efficiency when NADH was the coenzyme. Both K21A and K21A/N272D preferred NADH to NADPH, their catalytic efficiencies for NADPH were almost zero. The catalytic efficiency of K21A/N272D for NADH was almost 9-fold and 2-fold that of K21A and the wild-type enzyme, respectively. Complete reversal of coenzyme specificity toward NADH and improved catalytic efficiency were achieved. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Qi-Kai Zeng, Hong-Li Du, Jing-Fang Wang have contributed equally to this work.     

Lysine 21突变对树干毕赤氏酵母木糖还原酶辅酶依赖性的影响

木糖还原酶是重组酿酒酵母工程菌利用木糖生成乙醇代谢途径中的关键酶, 该关键酶在利用木糖时依赖NADPH而不是NADH是导致酿酒酵母代谢木糖生成乙醇的最终产率低的主要原因之一。为了改变树干毕赤氏酵母木糖还原酶的辅酶依赖性, 对它的第21位赖基酸Lys进行了突变。利用质粒载体pET28b分别将突变后的基因K21A-XYL1、K21R-XYL1及野生基因WT-XYL1在大肠杆菌E. coli BL21(DE3)中进行表达, 表达后的蛋白经His-Tag纯化柱纯化后测定酶学性质。结果表明: K21R突变子的辅酶依赖性没有改变, 但K21A突变子的辅酶依赖性由NADPH完全逆转为NADH。     

Identification of lysine-78 as an essential residue in the Saccharomyces cerevisiae xylose reductase

Yeast xylose reductases are hypothesized as hybrid enzymes as their primary sequences contain elements of both the aldo-keto reductases (AKR) and short chain dehydrogenase/reductase (SDR) enzyme families. During catalysis by members of both enzyme families, an essential Lys residue H-bonds to a Tyr residue that donates proton to the aldehyde substrate. In the Saccharomyces cerevisiae xylose reductase, Tyr49 has been identified as the proton donor. However, the primary sequence of the enzyme contains two Lys residues, Lys53 and Lys78, corresponding to the conserved motifs for SDR and AKR enzyme families, respectively, that may H-bond to Tyr49. We used site-directed mutagenesis to substitute each of these Lys residues with Met. The activity of the K53M variant was slightly decreased as compared to the wild-type, while that of the K78M variant was negligible. The results suggest that Lys78 is the essential residue that H-bonds to Tyr49 during catalysis and indicate that the active site residues of yeast xylose reductases match those of the AKR, rather than SDR, enzymes. Intrinsic enzyme fluorescence spectroscopic analysis suggests that Lys78 may also contribute to the efficient binding of NADPH to the enzyme.     

Altering coenzyme specificity of <Emphasis Type="Italic">Pichia stipitis</Emphasis> xylose reductase by the semi-rational approach CASTing

Background  

The NAD(P)H-dependent Pichia stipitis xylose reductase (PsXR) is one of the key enzymes for xylose fermentation, and has been cloned into the commonly used ethanol-producing yeast Saccharomyces cerevisiae. In order to eliminate the redox imbalance resulting from the preference of this enzyme toward NADPH, efforts have been made to alter the coenzyme specificity of PsXR by site-directed mutagenesis, with limited success. Given the industrial importance of PsXR, it is of interest to investigate further ways to create mutants of PsXR that prefers NADH rather than NADPH, by the alternative directed evolution approach.     

A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis

Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD⁺ to NADP⁺. In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH.     

Xylose reductase from the thermophilic fungus Talaromyces emersonii: cloning and heterologous expression of the native gene (Texr) and a double mutant (TexrK271R + N273D) with altered coenzyme specificity

Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene encoding xylose reductase (Texr) was isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli and purified to homogeneity. Texr encodes a 320 amino acid protein with a molecular weight of 36 kDa, which exhibited high sequence identity with other xylose reductase sequences and was shown to be a member of the aldoketoreductase (AKR) superfamily with a preference for reduced nicotinamide adenine dinucleotide phosphate (NADPH) as coenzyme. Given the potential application of xylose reductase enzymes that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation of five carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed mutagenesis. The TeXR^K271R+N273D double mutant displayed an altered coenzyme preference with a 16-fold improvement in NADH utilization relative to the wild type and therefore has the potential to reduce redox imbalance of xylose fermentation in recombinant S. cerevisiae strains. Expression of Texr was shown to be inducible by the same carbon sources responsible for the induction of genes encoding enzymes relevant to lignocellulose hydrolysis, suggesting a coordinated expression of intracellular and extracellular enzymes relevant to hydrolysis and metabolism of pentose sugars in T. emersonii in adaptation to its natural habitat. This indicates a potential advantage in survival and response to a nutrient-poor environment.     

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

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-274→Arg and Arg-280→His 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.     

Properties of NAD (P) H-linked aldose reductase from Crytococcus lactativorus

A yeast growing at 48°C was isolated from soil and the strain was identified as Cryptococcus lactativorus. The aldose reductase which the strain produced was purified 114-fold with an overall recovery of 36%. The stability of the enzyme was higher than that of other aldose reductases. The half life of the enzyme was 800 h and 14 h at 30°C and 50°C, respectively. The enzyme showed the best activity with d-xylose. l-Sorbose and d-fructose were also reduced by the enzyme. The enzyme was active with both NADPH and NADH as a conenzyme, and the activity with NADH was 1.25 times higher than that with NADPH. The K_m^app value for d-xylose was 8.6 mM and the V_max^app was 20.8 units/mg NADH was used as a coenzyme. The K_m^app values for NADPH and NADH were 6μM and 170 μM, respectively, when d-glucose was used as a substrate.     

Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD⁺-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys²⁷⁴→Arg Asn²⁷⁶→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.     

Identifying determinants of NADPH specificity in Baeyer-Villiger monooxygenases.

The Baeyer-Villiger monooxygenase (BVMO), 4-hydroxyacetophenone monooxygenase (HAPMO), uses NADPH and O(2) to oxidize a variety of aromatic ketones and sulfides. The FAD-containing enzyme has a 700-fold preference for NADPH over NADH. Sequence alignment with other BVMOs, which are all known to be selective for NADPH, revealed three conserved basic residues, which could account for the observed coenzyme specificity. The corresponding residues in HAPMO (Arg339, Lys439 and Arg440) were mutated and the properties of the purified mutant enzymes were studied. For Arg440 no involvement in coenzyme recognition could be shown as mutant R440A was totally inactive. Although this mutant could still be fully reduced by NADPH, no oxygenation occurred, indicating that this residue is crucial for completing the catalytic cycle of HAPMO. Characterization of several Arg339 and Lys439 mutants revealed that these residues are indeed both involved in coenzyme recognition. Mutant R339A showed a largely decreased affinity for NADPH, as judged from kinetic analysis and binding experiments. Replacing Arg339 also resulted in a decreased catalytic efficiency with NADH. Mutant K439A displayed a 100-fold decrease in catalytic efficiency with NADPH, mainly caused by an increased K(m). However, the efficiency with NADH increased fourfold. Saturation mutagenesis at position 439 showed that the presence of an asparagine or a phenylalanine improves the catalytic efficiency with NADH by a factor of 6 to 7. All Lys439 mutants displayed a lower affinity for AADP(+), confirming a role of the lysine in recognizing the 2'-phosphate of NADPH. The results obtained could be extrapolated to the sequence-related cyclohexanone monooxygenase. Replacing Lys326 in this BVMO, which is analogous to Lys439 in HAPMO, again changed the coenzyme specificity towards NADH. These results indicate that the strict NADPH dependency of this class of monooxygenases is based upon recognition of the coenzyme by several basic residues.     

Structural and functional properties of aldose xylose reductase from the D-xylose-metabolizing yeast Candida tenuis

The primary structure of the aldose xylose reductase from Candida tenuis (CtAR) is shown to be 39% identical to that of human aldose reductase (hAR). The catalytic tetrad of hAR is completely conserved in CtAR (Tyr51, Lys80, Asp46, His113). The amino acid residues involved in binding of NADPH by hAR (D.K. Wilson, et al., Science 257 (1992) 81-84) are 64% identical in CtAR. Like hAR the yeast enzyme is specific for transferring the 4-pro-R hydrogen of the coenzyme. These properties suggest that CtAR is a member of the aldo/keto reductase superfamily. Unlike hAR the enzyme from C. tenuis has a dual coenzyme specificity and shows similar specificity constants for NADPH and NADH. It binds NADP(+) approximately 250 times less tightly than hAR. Typical turnover numbers for aldehyde reduction by CtAR (15-20 s(-1)) are up to 100-fold higher than corresponding values for hAR, probably reflecting an overall faster dissociation of NAD(P)(+) in the reaction catalyzed by the yeast enzyme.     

Investigation of the Role of a Conserved Glycine Motif in the Saccharomyces cerevisiae Xylose Reductase

All yeast xylose reductases, with the exception of that from Schizosaccharomyces pombe, possess the catalytic and coenzyme-binding elements from both the aldo–keto reductase and short-chain dehydrogenase–reductase (SDR) enzyme families in their primary sequences. In the Saccharomyces cerevisiae xylose reductase (XR), the SDR-like coenzyme-binding GXXXGXG motif (Gly motif) is located between residues 128 and 134, with the third Gly residue being replaced by an Asp. We used site-directed mutagenesis to study the role of this SDR-like Gly motif in the S. cerevisiae xylose reductase. Site-directed mutagenesis of the individual conserved Gly residue positions (G128A, G132A, D134G, and D134A) did not significantly affect the specific activity, kinetic constants (K_m, K_cat, and K_cat/K_m), or dissociation constants (K_d) in any of the variants compared with the wild type. Deletion of the entire Gly motif produced an unstable protein that could not be purified. These results indicate that the SDR-like Gly motif likely provides support to the overall structure of the enzyme, but it does not contribute directly to coenzyme binding in this XR.     

Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis.

Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography. The enzyme was active with various aldose substrates, such as DL-glyceraldehyde, L-arabinose, D-xylose, D-ribose, D-galactose and D-glucose. Hence the xylose reductase of Pichia stipitis is an aldose reductase (EC 1.1.1.21). Unlike all aldose reductases characterized so far, the enzyme from this yeast was active with both NADPH and NADH as coenzyme. The activity with NADH was approx. 70% of that with NADPH for the various aldose substrates. NADP+ was a potent inhibitor of both the NADPH- and NADH-linked xylose reduction, whereas NAD+ showed strong inhibition only with the NADH-linked reaction. These results are discussed in the context of the possible use of Pichia stipitis and similar yeasts for the anaerobic conversion of xylose into ethanol.     

Isolation of a sn-glycerol 3-phosphate: NAD oxidoreductase from spinach leaves.

An NAD-dependent glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD oxidoreductase; EC 1.1.1.8) has been purified from spinach leaves by a three-step procedure involving ion-exchange, gel filtration, and affinity chromatography. The enzyme has been purified over 10,000-fold to a specific activity of 38. It has a molecular weight of approximately 63,500. The pH optimum for the reduction of dihydroxyacetone phosphate is 6.8 and for glycerol 3-phosphate oxidation it is 9.5. During dihydroxyacetone phosphate reduction hyperbolic kinetics were observed when either NADH or dihydroxyacetone phosphate was the variable substrate, but concentrations of NADH greater than 150 μm were inhibitory. Michaelis constants were 0.30–0.35 mm for dihydroxyacetone phosphate and 0.01 mm for NADH. Glycerol 3-phosphate oxidation obeyed Michaelis-Menten kinetics with a K_m of 0.19 mm for NAD and 1.6 mm for glycerol 3-phosphate. The enzyme was specific for those substrates, and dihydroxyacetone, glyceraldehyde, glyceraldehyde 3-phosphate, NADPH, NADP, and glycerol were not utilized. The spinach leaf enzyme appears to be in the cytoplasm and probably functions for the production of glycerol 3-phosphate from dihydroxyacetone phosphate.     

Improving ethanol and xylitol fermentation at elevated temperature through substitution of xylose reductase in Kluyveromyces marxianus

Thermo-tolerant yeast Kluyveromyces marxianus is able to utilize a wide range of substrates, including xylose; however, the xylose fermentation ability is weak because of the redox imbalance under oxygen-limited conditions. Alleviating the intracellular redox imbalance through engineering the coenzyme specificity of NADPH-preferring xylose reductase (XR) and improving the expression of XR should promote xylose consumption and fermentation. In this study, the native xylose reductase gene (Kmxyl1) of the K. marxianus strain was substituted with XR or its mutant genes from Pichia stipitis (Scheffersomyces stipitis). The ability of the resultant recombinant strains to assimilate xylose to produce xylitol and ethanol at elevated temperature was greatly improved. The strain YZB014 expressing mutant PsXR N272D, which has a higher activity with both NADPH and NADH as the coenzyme, achieved the best results, and produced 3.55 g l^?1 ethanol and 11.32 g l^?1 xylitol—an increase of 12.24- and 2.70-fold in product at 42 °C, respectively. A 3.94-fold increase of xylose consumption was observed compared with the K. marxianus YHJ010 harboring KmXyl1. However, the strain YZB015 expressing a mutant PsXR K21A/N272D, with which co-enzyme preference was completely reversed from NADPH to NADH, failed to ferment due to the low expression. So in order to improve xylose consumption and fermentation in K. marxianus, both higher activity and co-enzyme specificity change are necessary.     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

毕赤酵母木糖还原酶定点突变改善其对双辅酶的亲和力

通过毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase, XR)基因定点突变,获得NADH高亲和力的毕赤酵母木糖还原酶(PsXR),改善了辅酶不同而导致的酿酒酵母胞内氧化还原失衡.同时克隆了PsXR编码基因,通过BLAST工具进行同源性搜索,并用生物软件进行序列比对和结构分析,确定突变位点.用融合PCR方法进行定点突变,并在大肠杆菌表达系统中进行融合表达,且对表达产物进行HIS-TAG亲和纯化,分光光度法检测酶活性,计算比活力.本研究成功获得突变XR编码基因,并收集了纯化的突变蛋白.酶活性检测和比活力计算显示,3种突变酶对2种辅酶的亲和力在一定程度上都发生了变化.与未突变的PsXR相比,3种突变酶对辅酶NADPH的亲和力均显著下降,突变酶M3对辅酶NADH的亲和力未发生变化,而突变酶M1和M4对辅酶NADH的亲和力显著升高,其中突变酶M1对NADH的亲和力明显提高,对NADPH的亲和力明显下降,其活性主要依赖辅酶NADH,提示K270R位点在XR与辅酶结合中起关键作用.     

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP⁺, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.     

Reduction of ascorbate free radical by the plasma membrane of synaptic terminals from rat brain

Synaptic plasma membranes (SPMV) decrease the steady state ascorbate free radical (AFR) concentration of 1 mM ascorbate in phosphate/EDTA buffer (pH 7), due to AFR recycling by redox coupling between ascorbate and the ubiquinone content of these membranes. In the presence of NADH, but not NADPH, SPMV catalyse a rapid recycling of AFR which further lower the AFR concentration below 0.05 μM. These results correlate with the nearly 10-fold higher NADH oxidase over NADPH oxidase activity of SPMV. SPMV has NADH-dependent coenzyme Q reductase activity. In the presence of ascorbate the stimulation of the NADH oxidase activity of SPMV by coenzyme Q₁ and cytochrome c can be accounted for by the increase of the AFR concentration generated by the redox pairs ascorbate/coenzyme Q₁ and ascorbate/cytochrome c. The NADH:AFR reductase activity makes a major contribution to the NADH oxidase activity of SPMV and decreases the steady-state AFR concentration well below the micromolar concentration range.     

Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed mutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214-->Ala and Arg214-->Gln, the effector role of substrate is lost. For enzyme Arg214-->Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214-->Lys fails to stabilize the 4 alpha-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214-->Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.