Phosphite dehydrogenase

The latter drawback was successfiiUy overcome by designing a double-mutant PTDH (Glul75Ala, Alal76Arg), which was capable of efficiently regenerating both CO factors. In fact, in comparison to the wild-type enzyme, the mutant showed a catalytic efficiency with NADP that was 1000-fold higher. In addition, 

Recently, one of the mutations involved in the cofactor specificity (Alal76Arg) has been incorporated in this thermostable mutant by site-directed mutagenesis [29]. The evaluation of the performance of this novel enzyme in NADPH regeneration was carried out using two synthetically relevant enzymatic reactions as model 

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Woodyer, R., van der Donk, W.A. and Zhao, H.M. (2006) Optimizing a biocatalyst for improved NAD(P)H regeneration directed evolution of phosphite dehydrogenase. Combinatorial Chemistry High Throughput Screening, 9, 237-245. 

Fig. 7 Xylose reductase catalyzed xylitol synthesis. The coenzyme NADPH was regenerated by a NADP+-accepting mutant of phosphite dehydrogenase...

In this particular case, we decided to take advantage of the favorable thermodynamic equilibrium constant that drives the oxidation of phosphite to phosphate mediated by a recently described phosphite dehydrogenase (PTDH) [117] to a nearly irreversible process [118]. The exquisite selectivity of PTDH for phosphite also precludes any side reaction that can occur in case, for example, an ADH is used. These characteristics render PTDH as an ideal candidate for use as a coenzyme regenerating enzyme (GRE) in combination with BVMOs or other NAD(P)H-dependent enzymes. 

A common aim of directed evolution is to increase the stability of an enzyme to conditions of practical use that may be very different from those the enzyme naturally functions in. Factors such as heat, altered pH, and the presence of oxidants or organic solvents can lead to denaturation or loss of enzyme function. Many researchers have successfully increased the stability of an enzyme to thermal denaturation (41, 42). Work with p-nitrobenzyl esterase uicreased the melting temperature 14°C after six rounds of EP-PCR and recombination without forfeiting enzyme activity (41). As another example, phosphite dehydrogenase catalyzes the formation of phosphate from phosphite, by reducing NAD+ to NADH. However, the usefulness... 

Johannes TW, Woodyer RD, Zhao H. Directed evolution of a thermostable phosphite dehydrogenase for NAD(P)H regeneration. 

More recently, phosphite dehydrogenase has been shown to offer a promising alternative [737, 738] The equilibrium is extremely favorable, both phosphite and phosphate are inoccuous to enzymes and act as buffer. The wild-type enzyme from Pseudomonas stutzeri accepts ony NAD" [739], but thermostable mutants were generated which are also able to reduce NADP" [740-742]. 

Relyea HA, van der Donk WA (2005) Mechanism and applications of phosphite dehydrogenase. BioorgChem 33 171-189... 

Vitis JM, White AK, Metcalf WW, van der Donk WA (2002) Phosphite dehydrogenase a versatile cofactor-regeneration enzyme. Angew Chem Int Ed Engl 41 3257-3259... 

Woodyer R, van der Donk WA, Zhao H (2003) Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design. Biochemistry 42 11604-11614... 

This self-sufficient two-in-one redox biocatalyst enables the use of phosphite (HP03 ) as an inexpensive and sacrificial electron donor, and does not require an additional catalytic entity for coenzyme recycling. As model BVMOs, PAMO, CHMO, and CPMO were selected. No inhibition of the activity of either the BVMO or phosphite dehydrogenase (PTDH) by the substrate or product of the other subunit could be observed. 

Scheme 2.1 Practically irreversible oxidation of inorganic phosphite (hydrogen phospho-nate) to phosphate catalyzed by NADIPj -dependent phosphite dehydrogenase (PTDH).

Scheme 2.2 NADPH-dependent Baeyer-Villiger monooxygenases (BVMO) fused to a phosphite dehydrogenase (PTDH) for self-sufficient coenzyme regeneration.

Johannes, T.W., Woodyer, R.D., and Zhao, H. (2007) Efficient regeneration of NADPH using an engineered phosphite dehydrogenase. Biotechnol. Bioeng., 96, 18-26. 

Zhao, H., and Nair, S.K. (2012) Crystal stmctures of phosphite dehydrogenase provide insights into nicotinamide... 

Scheme 8.7 Phosphite dehydrogenase (PDH)-catalyzed regeneration of NADPH to promote the reduction of xylose catalyzed by xylose reductase.

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