Cloning, Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from Pseudomonas sp. K in Escherichia coli

Liu, Danfeng; Ding, Haitao; Du, Yiqing; Zhao, Yong; Jia, Xiaoming

doi:10.1007/s12010-011-9518-2

Cited by 4 publications

(3 citation statements)

References 32 publications

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“…Another phosphite dehydrogenase from Pseudomonas sp. K was reported while this paper was under review . Analysis of the kinetic parameters of these enzymes illustrates that they all have similar k cat values despite a high degree of sequence diversity (39–72% pairwise identity).…”

Section: Discussionmentioning

confidence: 96%

“…K was reported while this paper was under review. 25 Analysis of the kinetic parameters of these enzymes illustrates that they all have similar k cat values despite a high degree of sequence diversity (39–72% pairwise identity). The reaction catalyzed by PTDHs has a very strong thermodynamic driving force, and it has been suggested that perhaps the rather moderate values of k cat were the result of a “young” enzyme that had relatively recently evolved from a progenitor in the DHDH family.…”

Section: Discussionmentioning

confidence: 99%

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Investigation of the Role of Arg301 Identified in the X-ray Structure of Phosphite Dehydrogenase

et al. 2012

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Phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri catalyzes the nicotinamide adenine dinucleotide-dependent oxidation of phosphite to phosphate. The enzyme belongs to the family of d-hydroxy acid dehydrogenases (DHDHs). A search of the protein databases uncovered many additional putative phosphite dehydrogenases. The genes encoding four diverse candidates were cloned and expressed, and the enzymes were purified and characterized. All oxidized phosphite to phosphate and had similar kinetic parameters despite a low level of pairwise sequence identity (39–72%). A recent crystal structure identified Arg301 as a residue in the active site that has not been investigated previously. Arg301 is fully conserved in the enzymes shown here to be PTDHs, but the residue is not conserved in other DHDHs. Kinetic analysis of site-directed mutants of this residue shows that it is important for efficient catalysis, with an ∼100-fold decrease in kcat and an almost 700-fold increase in Km,phosphite for the R301A mutant. Interestingly, the R301K mutant displayed a slightly higher kcat than the parent PTDH, and a more modest increase in Km for phosphite (nearly 40-fold). Given these results, Arg301 may be involved in the binding and orientation of the phosphite substrate and/or play a catalytic role via electrostatic interactions. Three other residues in the active site region that are conserved in the PTDH orthologs but not DHDHs were identified (Trp134, Tyr139, and Ser295). The importance of these residues was also investigated by site-directed mutagenesis. All of the mutants had kcat values similar to that of the wild-type enzyme, indicating these residues are not important for catalysis.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Investigation of the Role of Arg301 Identified in the X-ray Structure of Phosphite Dehydrogenase

et al. 2012

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“…Recent reports have shown that a variety of organisms encode enzymes with PTDH activity [13] – [16] . In addition, the crystal structure of the thermostable PTDH mutant was recently published [17] .…”

Section: Introductionmentioning

confidence: 99%

Chemical Rescue and Inhibition Studies to Determine the Role of Arg301 in Phosphite Dehydrogenase

et al. 2014

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Phosphite dehydrogenase (PTDH) catalyzes the NAD+-dependent oxidation of phosphite to phosphate. This reaction requires the deprotonation of a water nucleophile for attack on phosphite. A crystal structure was recently solved that identified Arg301 as a potential base given its proximity and orientation to the substrates and a water molecule within the active site. Mutants of this residue showed its importance for efficient catalysis, with about a 100-fold loss in k cat and substantially increased K m,phosphite for the Ala mutant (R301A). The 2.35 Å resolution crystal structure of the R301A mutant with NAD+ bound shows that removal of the guanidine group renders the active site solvent exposed, suggesting the possibility of chemical rescue of activity. We show that the catalytic activity of this mutant is restored to near wild-type levels by the addition of exogenous guanidinium analogues; Brønsted analysis of the rates of chemical rescue suggests that protonation of the rescue reagent is complete in the transition state of the rate-limiting step. Kinetic isotope effects on the reaction in the presence of rescue agents show that hydride transfer remains at least partially rate-limiting, and inhibition experiments show that K i of sulfite with R301A is ∼400-fold increased compared to the parent enzyme, similar to the increase in K m for phosphite in this mutant. The results of our experiments indicate that Arg301 plays an important role in phosphite binding as well as catalysis, but that it is not likely to act as an active site base.

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Significant improvement of thermal stability of glucose 1-dehydrogenase by introducing disulfide bonds at the tetramer interface

Ding

Gao

Liu

et al. 2013

Enzyme and Microbial Technology

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Cloning, Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from Pseudomonas sp. K in Escherichia coli

Cited by 4 publications

References 32 publications

Investigation of the Role of Arg301 Identified in the X-ray Structure of Phosphite Dehydrogenase

Investigation of the Role of Arg301 Identified in the X-ray Structure of Phosphite Dehydrogenase

Chemical Rescue and Inhibition Studies to Determine the Role of Arg301 in Phosphite Dehydrogenase

Significant improvement of thermal stability of glucose 1-dehydrogenase by introducing disulfide bonds at the tetramer interface

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