Phosphite Dehydrogenase: A Versatile Cofactor-Regeneration Enzyme

Vrtis, Jennifer M.; White, Andrea K.; Metcalf, William W.; Donk, Wilfred A. van der

doi:10.1002/1521-3773(20020902)41:17<3257::aid-anie3257>3.0.co;2-n

Cited by 127 publications

(69 citation statements)

References 16 publications

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“…Finally, activity was reduced only 9 or 37% in the presence of sodium phosphite at 50 and 200 mM, respectively. This result suggests that N. crassa XR is compatible with phosphite dehydrogenase, a potentially efficient in situ NADPH regeneration system (31,32).…”

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confidence: 76%

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

Woodyer¹,

Simurdiak

Donk³

et al. 2005

Appl Environ Microbiol

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confidence: 76%

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

Woodyer¹,

Simurdiak

Donk³

et al. 2005

Appl Environ Microbiol

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“…This enzyme catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD ϩ to NADH. The kinetic and practical advantages of using PTDH for NADH regeneration have been recently reported (22).In addition to NADH regeneration, the enzyme PTDH also has considerable potential in regenerating NADPH. The cost of NADPH is significantly higher than that of NADH, and no widely used NADPH regeneration system is currently available.…”

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confidence: 99%

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confidence: 99%

Directed Evolution of a Thermostable Phosphite Dehydrogenase for NAD(P)H Regeneration

Johannes

Woodyer²,

Zhao

2005

Appl Environ Microbiol

142

106

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NAD(P)H-dependent oxidoreductases are valuable tools for synthesis of chiral compounds. The expense of the cofactors, however, requires in situ cofactor regeneration for preparative applications. We have attempted to develop an enzymatic system based on phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri to regenerate the reduced nicotinamide cofactors NADH and NADPH. Here we report the use of directed evolution to address one of the main limitations with the wild-type PTDH enzyme, its low stability. After three rounds of random mutagenesis and high-throughput screening, 12 thermostabilizing amino acid substitutions were identified. These 12 mutations were combined by site-directed mutagenesis, resulting in a mutant whose T 50 is 20°C higher and half-life of thermal inactivation at 45°C is >7,000-fold greater than that of the parent PTDH. The engineered PTDH has a half-life at 50°C that is 2.4-fold greater than the Candida boidinii formate dehydrogenase, an enzyme widely used for NADH regeneration. In addition, its catalytic efficiency is slightly higher than that of the parent PTDH. Various mechanisms of thermostabilization were identified using molecular modeling. The improved stability and effectiveness of the final mutant were shown using the industrially important bioconversion of trimethylpyruvate to L-tert-leucine. The engineered PTDH will be useful in NAD(P)H regeneration for industrial biocatalysis.The potential of enzyme-catalyzed transformations in the pharmaceutical and fine-chemical industries has long been considered to have great promise (7,10,23). Despite the development of rapid and efficient methods for creating designer biocatalysts, inherent limitations in the chemistry that these enzymes perform still persist. The majority of biocatalysts used in industry are hydrolytic in action (ϳ65%) and perform rather simple chemistry (4). More complicated enzymatic reactions often require one or more costly cofactors, which, when added in stoichiometric quantities, make the process not economically feasible. Oxidoreductases, for example, catalyze a myriad of regio-, chemo-, and stereospecific reactions on a variety of functional groups, but often require either NADH or NADPH as a cofactor (8, 11).Various in situ regeneration methods have been developed to allow the use of catalytic quantities of NAD(P) ϩ and NAD(P)H (20). The most successful and widely used enzymatic NADH regeneration system is based on the Candida boidinii formate dehydrogenase (FDH) (1). We have attempted to develop an NADH regeneration process using the enzyme phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri (2). This enzyme catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD ϩ to NADH. The kinetic and practical advantages of using PTDH for NADH regeneration have been recently reported (22).In addition to NADH regeneration, the enzyme PTDH also has considerable potential in regenerating NADPH. The cost of NADPH is significantly higher than that of NADH, and no widel...

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“…The following two enzymes catalyze these reactions: 2-oxoglutarate͞hypophosphite dioxygenase catalyzes the oxidation of hypo-Pt to Pt (10), and Pt͞NAD oxidoreductase catalyzes the oxidation of Pt to phosphate (11). The latter reaction is the most thermodynamically favorable reduction of NAD that is known, and this trait makes this enzyme particularly useful as a cofactor-regenerating catalyst for enzyme-based synthetic strategies (12). P biochemistry can be found also in more familiar organisms, such as Escherichia coli.…”

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A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase

Yang

Metcalf

2004

Proc. Natl. Acad. Sci. U.S.A.

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Genetic analysis indicates that Escherichia coli possesses two independent pathways for oxidation of phosphite (Pt) to phosphate. One pathway depends on the 14-gene phn operon, which encodes the enzyme C-P lyase. The other pathway depends on the phoA locus, which encodes bacterial alkaline phosphatase (BAP). Transposon mutagenesis studies strongly suggest that BAP is the only enzyme involved in the phoA-dependent pathway. This conclusion is supported by purification and biochemical characterization of the Pt-oxidizing enzyme, which was proven to be BAP by N terminus protein sequencing. Highly purified BAP catalyzed Pt oxidation with specific activities of 62-242 milliunits͞mg and phosphate ester hydrolysis with specific activities of 41-61 units͞ mg. Surprisingly, BAP catalyzes the oxidation of Pt to phosphate and molecular H 2. Thus, BAP is a unique Pt-dependent, H2-evolving hydrogenase. This reaction is unprecedented in both P and H biochemistry, and it is likely to involve direct transfer of hydride from the substrate to water-derived protons.U nlike the other major elements in living organisms, P is commonly considered to be a redox conservative element. Accordingly, the P centers found in the vast majority of biological intermediates, including inorganic phosphate (P i ), organic phosphate esters, and phosphoanhydrides, are fully oxidized (ϩ5 valence state). Nevertheless, it is now clear, although not widely appreciated, that many organisms are capable of metabolizing reduced P compounds, indicating that P redox reactions are biochemically possible. A wide array of prokaryotic (and some eukaryotic) organisms can synthesize or degrade reduced P compounds (1, 2). Moreover, the widespread occurrence of this trait strongly suggests that a selective advantage is conferred on organisms with the ability to metabolize reduced P compounds. Examination of reduced P metabolism has revealed a wealth of unusual biology and biochemistry. The bacterium Desulfotignum phosphitoxidans gains energy for growth by oxidation of phosphite (Pt) coupled to either sulfate reduction or acetogenesis while fixing CO 2 as its sole C source (3, 4). Many other organisms can use reduced P compounds as sole P sources (5-8). For example, Pseudomonas stutzeri is capable of oxidizing hypophosphite (P valence, ϩ1) to phosphate by means of a Pt (P valence, ϩ3) intermediate (9). The following two enzymes catalyze these reactions: 2-oxoglutarate͞hypophosphite dioxygenase catalyzes the oxidation of hypo-Pt to Pt (10), and Pt͞NAD oxidoreductase catalyzes the oxidation of Pt to phosphate (11). The latter reaction is the most thermodynamically favorable reduction of NAD that is known, and this trait makes this enzyme particularly useful as a cofactor-regenerating catalyst for enzyme-based synthetic strategies (12). P biochemistry can be found also in more familiar organisms, such as Escherichia coli. As shown below, E. coli has two pathways for the oxidation of Pt. Our examination of these pathways demonstrates that thoroughly characterized biochemist...

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Phosphite Dehydrogenase: A Versatile Cofactor-Regeneration Enzyme

Cited by 127 publications

References 16 publications

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

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

Directed Evolution of a Thermostable Phosphite Dehydrogenase for NAD(P)H Regeneration

A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase

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