The enzyme phosphite dehydrogenase (PTDH) catalyzes the NAD+-dependent conversion of phosphite to phosphate and represents the first biological catalyst that has been characterized to carry out the enzymatic oxidation of phosphorus. Despite over a decade’s worth of investigation into both the mechanism of its unusual reaction, as well as its utility in cofactor regeneration, there has been a lack of any structural data on PTDH. Here we present the co-crystal structure of an engineered thermostable variant of PTDH bound to NAD+ (1.7 Å resolution), as well as four other co-crystal structures of thermostable PTDH and its variants with different ligands (all between 1.85 – 2.3 Å resolution). These structures provide a molecular framework for understanding prior mutational analysis, and point to additional residues, located in the active site, that may contribute to the enzymatic activity of this highly unusual catalyst.
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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