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Klimacek, Mario; Eis, Christian; Nidetzky, Bernd

doi:10.1023/a:1008986623007

Cited by 6 publications

(6 citation statements)

References 10 publications

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“…Chemicals and other materials were described elsewhere (4,(12)(13)(14). Reported methods were used for preparation of wildtype PfM2DH (18).…”

Section: Methodsmentioning

confidence: 99%

“…In the shown primers, the underlined codons produce the respective sitedirected substitution, and the mismatched bases are indicated in bold. Plasmid pETR241-PfM2DH encoding C-terminally His-tagged wild-type enzyme was used as template for PCR according to a reported protocol (12). To obtain the gene encoding N191D-N300D, we subjected the plasmids for N191D and N300D to a double digestion by NcoI and BamHI.…”

Section: Methodsmentioning

confidence: 99%

“…Kinetic Characterization-Methods for steady-state kinetic characterization were described in previous reports (12)(13)(14)18). The buffers used (each 100 mM) were MES/NaOH (pH 5.2-6.8), Tris/HCl (pH 7.1-9.0), and glycine/NaOH (pH 9.0 - (19).…”

Section: Preparation Of N191d N300d and N191d-n300d-inversementioning

confidence: 99%

“…Mutational analysis has delineated catalytic roles for Asn-191 and Asn-300 during NAD(H)-dependent interconversion of mannitol and fructose by PfM2DH (4,12). Positioning effects and electrostatic stabilization are exploited to facilitate formation of the reactive conformers in each direction of the reaction and to provide selective rate enhancement to the hydride transfer step.…”

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confidence: 99%

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From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign

Klimacek

Nidetzky

2010

Journal of Biological Chemistry

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Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD ؉ -mannitol (K int ) was altered dramatically (10 4 -to 10 5 -fold) from being balanced in the wild-type enzyme (K int ≈ 3) to favoring enzyme-NAD ؉ -mannitol in the single site mutants, N191D and N300D.The change in K int reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn 3 Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD ؉ -mannitol complex.N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ؎ 0.1 and 6.3 ؎ 0.2, respectively. The mutant incorporating both Asn3 Asp substitutions behaved as a slow "fructose reductase" at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8 -10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively "one-way" reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis.Enzymatic NAD or NADP-dependent interconversion of alcohol and carbonyl groups is a key chemical transformation in biology. The thermodynamic equilibrium constant of the reaction usually favors alcohol under a wide range of external conditions. However, the directional preference of biological catalysis, expressed as the ratio of reaction rate constants in direction of carbonyl reduction (k R ) and alcohol oxidation (k O ), is not uniform among natural enzymes and underpins a widely used classification that considers "alcohol dehydrogenases" (k R /k O Յ 1) and "carbonyl reductases" (k R /k O ӷ 1). Horse liver alcohol dehydrogenase and human aldose reductase are representative members of each class, having k R /k O values of ϳ1 (1) and 217 (2), respectively. Work on lactate dehydrogenase has shown that k R /k O is sensitive to active-site structural changes resulting from site-directed mutagenesis (3). However, a clear mechanistic basis for the distinction between dehydrogenases and reductases is currently lacking.The oxyanion binding pocket of PfM2DH (Pseudomonas fluorescens ma...

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“…Chemicals and other materials were described elsewhere (4,(12)(13)(14). Reported methods were used for preparation of wildtype PfM2DH (18).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Preparation Of N191d N300d and N191d-n300d-inversementioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign

Klimacek

Nidetzky

2010

Journal of Biological Chemistry

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“…Thirdly, other bacterial MtDHs belong to a polyol-specific long-chain dehydrogenase group (Schneider et al, 1993). These long-chain MtDHs are often monomeric; they are characterized by the catalytic consensus motif Lys-Xaa(4/5)-Asn-Xaa(2)-His and do not require metals for catalysis (Kavanagh et al, 2002a,b;Klimacek & Nidetzky, 2002). The X-ray structures of Agaricus bisporus MtDH (a short-chain dehydrogenase; Hö rer et al, 2001) and of Pseudomonas fluorescens MtDH (a long-chain dehydrogenase; Kavanagh et al, 2002a,b) are known.…”

Section: Introductionmentioning

confidence: 99%

Crystallization, preliminary X-ray diffraction and structure analysis ofThermotoga maritimamannitol dehydrogenase

et al. 2007

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Trehalose production at high temperature exploiting an immobilized cell bioreactor

et al. 2002

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The enzymatic production of trehalose from dextrins was studied as a series reaction in a packed bed reactor containing immobilized recombinant Escherichia coli cells, expressing either the Sulfolobus solfataricus (strain MT4) trehalosyl-dextrin forming enzyme (TDFE) or the trehalose-forming enzyme (TFE). The cells, subjected to thermal treatments to increase cell permeability and to inactivate the unwanted host proteins, were entrapped separately or together in a calcium alginate polymeric matrix. The biocatalyst beads were used to pack a tubular glass reactor that was operated in a recycle mode. The performances of a bioreactor containing alternate layers of EcTFE and EcTDFE alginate beads were evaluated and compared with the performance of the co-immobilized biocatalysts. The latter showed a superior throughput, therefore the bioreactor packed with the co-entrapped biocatalysts was tested for the production of trehalose from concentrated dextrin solutions (10%-30% w/v) and a conversion up to 90% was obtained. This conversion corresponded to a production of 127 g trehalose h(-1) kg(-1) of biocatalyst. The results obtained suggest that the bioprocess described may be of interest in the development of a large-scale industrial process for trehalose production at high temperature.

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Cited by 6 publications

References 10 publications

From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign

From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign

Crystallization, preliminary X-ray diffraction and structure analysis ofThermotoga maritimamannitol dehydrogenase

Trehalose production at high temperature exploiting an immobilized cell bioreactor

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