Kinetic characterization of d-xylose isomerases by enzymatic assays using d-sorbitol dehydrogenase

Kersters-Hilderson, H; Callens, Mia; Opstal, Omer Van; Vangrysperre, W; Bruyne, Clement K. De

doi:10.1016/0141-0229(87)90067-6

Cited by 38 publications

(22 citation statements)

References 36 publications

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“…Values of k cat = 3.3 s −1 and K m = 5.0 mM at pD 8.0 were determined for this isomerization reaction under otherwise similar conditions. By comparison, the XI-catalyzed ( Streptomyces violaceoruber ) isomerization of D-xylose in H 2 O at 35 °C exhibits a broad pH optimum at neutral pH, with k cat = 10 s −1 and K m = 2.8 mM (23). …”

Section: Resultsmentioning

confidence: 99%

“…Typically, a 3 – 4-fold volume excess of dialysis buffer over sample was used, and the buffer was changed 7 times. The concentration of XI in stock solutions used for kinetic and product studies was determined from the absorbance at 280 nm using ε = 0.96 mg −1 ml cm −1 (30) and a molecular weight of 43,000 Dalton/monomer (23). The concentration of XI in stock solutions used for crystallization studies was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA).…”

Section: Methodsmentioning

confidence: 99%

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Binding Energy and Catalysis by d-Xylose Isomerase: Kinetic, Product, and X-ray Crystallographic Analysis of Enzyme-Catalyzed Isomerization of (R)-Glyceraldehyde

et al. 2011

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D-Xylose isomerase (XI) and triosephosphate isomerase (TIM) catalyze the aldose-ketose isomerization reactions of D-xylose and D-glyceraldehyde 3-phosphate (DGAP), respectively. D-Glyceraldehyde (DGA) is the triose fragment common to the substrates for XI and TIM. The XI-catalyzed isomerization of DGA to give dihydroxyacetone (DHA) in D2O was monitored by 1H NMR spectroscopy and kcat/Km = 0.034 M−1 s−1 was determined for this isomerization at pD 7.0. This is similar to kcat/Km = 0.017 M−1 s−1 for the TIM-catalyzed carbon deprotonation reaction of DGA in D2O at pD 7.0 [Amyes, T. L.; O’Donoghue, A. C. and Richard J. P. (2001) J. Am. Chem. Soc. 123, 11325–11326]. The much larger activation barrier for XI-catalyzed isomerization of D-xylose (kcat/Km = 490 M−1 s−1) than for the TIM-catalyzed isomerization of DGAP (kcat/Km = 9.6 x 106 M−1 s−1) is due to: (i) The larger barrier to conversion of cyclic D-xylose to the reactive linear sugar (5.4 kcal/mol) than for conversion of DGAP hydrate to the free aldehyde (1.7 kcal/mol). (ii) The smaller intrinsic binding energy [Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219–410] of the terminal ethylene glycol fragment of D-xylose (9.3 kcal/mol) than of the phosphodianion group of DGAP (ca. 12 kcal/mol). The XI-catalyzed isomerization of DGA in D2O at pD 7.0 gives a 90% yield of [1-1H]-DHA and a 10% yield of [1-2H]-DHA, the product of isomerization with deuterium incorporation from solvent D2O. By comparison, the transfer of 3H from labeled hexose substrate to solvent is observed only once in every 109 turnovers for the XI-catalyzed isomerization of [2-3H]-glucose in H2O [Allen, K. N., Lavie, A., Farber, G. K., Glasfeld, A., Petsko, G. A., and Ringe, D. (1994), Biochemistry 33, 1481–1487]. We propose that truncation of the terminal ethylene glycol fragment of D-xylose to give DGA results in a large decrease in the rate of XI-catalyzed isomerization with hydride transfer compared with that for proton transfer. An ultra-high resolution (0.97 Å) X-ray crystal structure was determined for the complex obtained by soaking crystals of XI with 50 mM DGA. The triose binds to XI as the unreactive hydrate, but ligand binding induces metal cofactor movement and conformational changes in active site residues similar to those observed for XI•sugar complexes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Binding Energy and Catalysis by d-Xylose Isomerase: Kinetic, Product, and X-ray Crystallographic Analysis of Enzyme-Catalyzed Isomerization of (R)-Glyceraldehyde

et al. 2011

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“…As demonstrated above, these mutant xylA genes were able to increase the growth rate of yeast cells on xylose; thus, we sought to understand the underlying mechanism of this improvement using in vitro kinetics assays. To do so, total protein was extracted from these strains, and enzyme assays were conducted using a spectrophotometry-based coupled-enzyme system (18). Similar to the growth rates, the enzyme activities of xylose isomerase mutants increased progressively with the rounds of mutagenesis and selection ( Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…Xylose isomerase activities from cell extracts were assayed by measuring the decrease of NADH in 1 ml of reaction mixture at 340 nm using a spectrophotometer (18). The reaction mixture contained 100 mM Tris-HCl buffer (pH 7.5), 0.15 mM NADH, 10 mM MgCl 2 , and 2 U sorbitol dehydrogenase (Roche, Mannheim, Germany).…”

Section: Methodsmentioning

confidence: 99%

Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

Lee

Jellison

Alper

2012

Appl Environ Microbiol

141

132

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The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

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“…The K m and k cat values were determined by a coupled enzymatic assay with D-sorbitol dehydrogenase (Boehringer Mannheim, Indianapolis, Ind.) (20). The reaction mixture contained 0.2 to 0.3 U of XI (1 U was defined as the amount of enzyme that converts 1 M D-xylose to D-xylulose/min), 1 U of D-sorbitol dehydrogenase (1 U was defined as the amount of enzyme that oxidizes 1 M NADH/min), 2 to 30 mM D-xylose, and 0.15 mM NADH (Boeringer Mannheim).…”

Section: Vol 70 2004 Dissolute Xylose Isomerase Recovery 4319mentioning

confidence: 99%

Restoration of a Defective Lactococcus lactis Xylose Isomerase

Park

Batt

2004

Appl Environ Microbiol

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The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl ؊ ) and IO-1 (Xyl ؉ ), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with K m and k cat being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The K m and k cat of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.

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Kinetic characterization of d-xylose isomerases by enzymatic assays using d-sorbitol dehydrogenase

Cited by 38 publications

References 36 publications

Binding Energy and Catalysis by d-Xylose Isomerase: Kinetic, Product, and X-ray Crystallographic Analysis of Enzyme-Catalyzed Isomerization of (R)-Glyceraldehyde

Binding Energy and Catalysis by d-Xylose Isomerase: Kinetic, Product, and X-ray Crystallographic Analysis of Enzyme-Catalyzed Isomerization of (R)-Glyceraldehyde

Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

Restoration of a Defective Lactococcus lactis Xylose Isomerase

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