Abstract
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.
Highlights
Phosphite dehydrogenase (PTDH), first discovered in Pseudomonas stutzeri WM88 [1], catalyzes the oxidation of phosphite to phosphate, which is coupled to the reduction of NAD+ to NADH
The addition of a saturating concentration of rescue agent lead to a 10- to 30-fold increase in krescue compared to kcat for the R301A mutant without any rescue reagent, yielding about 40% of wild-type activity with the most effective reagent, aminoguanidine (Table 1)
Previous mutagenesis studies demonstrated that Arg301 plays an important role in PTDH catalysis, but its precise role could not be elucidated [13]
Summary
Phosphite dehydrogenase (PTDH), first discovered in Pseudomonas stutzeri WM88 [1], catalyzes the oxidation of phosphite to phosphate, which is coupled to the reduction of NAD+ to NADH. The thermodynamics of the reaction are 15 kcal/mol exergonic [2], and PTDH has been adapted for use in cofactor regeneration strategies [3]. PTDH mutants have been engineered with increased thermostability [4] and relaxed cofactor specificity [5], resulting in a stable, highly active enzyme termed 17X-PTDH that is capable of regenerating NADPH in addition to NADH. Hydride transfer is fully rate-limiting in the reaction catalyzed by either wild-type PTDH or 17X-PTDH, as determined by kinetic isotope effects and pre-steady state experiments [11,12]. The enzyme follows an ordered kinetic mechanism with NAD+ binding before phosphite [1]. A number of questions about the chemical mechanism have emerged, including the identity of the catalytic base responsible for deprotonating the water nucleophile
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
