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Investigation of the role of Arg301 identified in the X-ray structure of phosphite dehydrogenase.

Hung JE, Fogle EJ, Christman HD, Johannes TW, Zhao H, Metcalf WW, van der Donk WA - Biochemistry (2012)

Bottom Line: Kinetic analysis of site-directed mutants of this residue shows that it is important for efficient catalysis, with an ~100-fold decrease in k(cat) and an almost 700-fold increase in K(m,phosphite) for the R301A mutant.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.All of the mutants had k(cat) values similar to that of the wild-type enzyme, indicating these residues are not important for catalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA.

ABSTRACT
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 k(cat) and an almost 700-fold increase in K(m,phosphite) for the R301A mutant. Interestingly, the R301K mutant displayed a slightly higher k(cat) than the parent PTDH, and a more modest increase in K(m) 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 k(cat) values similar to that of the wild-type enzyme, indicating these residues are not important for catalysis.

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Possible protonation states of phosphite and His292 in PTDH. Thetwo substrates bind to the enzyme in an ordered mechanism with NAD+ binding first.1 On the basis ofthe pH–rate profile, dianionic phosphite could bind to theNAD+–PTDH complex containing a protonated His292(blue). If this is the active complex, a residue other than His292must function as a base to deprotonate the water nucleophile. Alternatively,the pKa values of phosphite and the Hiscould be perturbed in the ternary complex such that a proton is transferredfrom the protonated His to the phosphite to generate a monoprotonatedphosphite as the actual electrophile (red). This form of the ternarycomplex could also be accessed if monoanionic phosphite binds to theNAD+–PTDH complex in which the His is unprotonated(reverse protonation based on their pKa values).
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fig2: Possible protonation states of phosphite and His292 in PTDH. Thetwo substrates bind to the enzyme in an ordered mechanism with NAD+ binding first.1 On the basis ofthe pH–rate profile, dianionic phosphite could bind to theNAD+–PTDH complex containing a protonated His292(blue). If this is the active complex, a residue other than His292must function as a base to deprotonate the water nucleophile. Alternatively,the pKa values of phosphite and the Hiscould be perturbed in the ternary complex such that a proton is transferredfrom the protonated His to the phosphite to generate a monoprotonatedphosphite as the actual electrophile (red). This form of the ternarycomplex could also be accessed if monoanionic phosphite binds to theNAD+–PTDH complex in which the His is unprotonated(reverse protonation based on their pKa values).

Mentions: For wt-PTDH, the pH–rate profile for kcat/Km,phosphite shows a bell-shapedcurve indicating that for optimal catalysis one group (on the proteinor the substrate) must be deprotonated and another group must be protonated.13 The pKa values deducedfrom the curve are 6.8 and 7.8, respectively. The lower pKa corresponds well to the second acid dissociation constantof phosphorous acid (the conjugate acid of phosphite), suggestingthat the enzyme may utilize the dianionic form of the substrate. Thehigher pKa would then correspond to anenzyme residue that must be protonated. One candidate would be His292(Figure 1), but if so, it could not be thecatalytic base that deprotonates the water nucleophile. One scenariothat would still allow His292 to serve as a base involves bindingof dianionic phosphite to PTDH with a protonated His292, followedby the transfer of a proton from His292 to phosphite in the ternarycomplex because of perturbed pKa valuesin the ternary complex (Figure 2). An alternativeinterpretation of the data is that phosphite must be protonated andHis292 deprotonated for substrate binding. In this interpretation,the minor, monoprotonated form of the substrate at the optimal pHof 7.25 would bind to the minor form of the enzyme with a deprotonatedHis (Figure 2). Such a reverse protonationscenario would result in an identical pH–rate profile becauseboth of these two protonation states of the substrate and enzyme wouldhave maximal occupancy at pH 7.25, decreasing at both higher and lowerpH values.13 Because the pKa values of the acidic and basic limbs of the PTDH pH–rateprofile are separated by <2 pH units, it is difficult to differentiatebetween normal and reverse protonation states.16


Investigation of the role of Arg301 identified in the X-ray structure of phosphite dehydrogenase.

Hung JE, Fogle EJ, Christman HD, Johannes TW, Zhao H, Metcalf WW, van der Donk WA - Biochemistry (2012)

Possible protonation states of phosphite and His292 in PTDH. Thetwo substrates bind to the enzyme in an ordered mechanism with NAD+ binding first.1 On the basis ofthe pH–rate profile, dianionic phosphite could bind to theNAD+–PTDH complex containing a protonated His292(blue). If this is the active complex, a residue other than His292must function as a base to deprotonate the water nucleophile. Alternatively,the pKa values of phosphite and the Hiscould be perturbed in the ternary complex such that a proton is transferredfrom the protonated His to the phosphite to generate a monoprotonatedphosphite as the actual electrophile (red). This form of the ternarycomplex could also be accessed if monoanionic phosphite binds to theNAD+–PTDH complex in which the His is unprotonated(reverse protonation based on their pKa values).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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fig2: Possible protonation states of phosphite and His292 in PTDH. Thetwo substrates bind to the enzyme in an ordered mechanism with NAD+ binding first.1 On the basis ofthe pH–rate profile, dianionic phosphite could bind to theNAD+–PTDH complex containing a protonated His292(blue). If this is the active complex, a residue other than His292must function as a base to deprotonate the water nucleophile. Alternatively,the pKa values of phosphite and the Hiscould be perturbed in the ternary complex such that a proton is transferredfrom the protonated His to the phosphite to generate a monoprotonatedphosphite as the actual electrophile (red). This form of the ternarycomplex could also be accessed if monoanionic phosphite binds to theNAD+–PTDH complex in which the His is unprotonated(reverse protonation based on their pKa values).
Mentions: For wt-PTDH, the pH–rate profile for kcat/Km,phosphite shows a bell-shapedcurve indicating that for optimal catalysis one group (on the proteinor the substrate) must be deprotonated and another group must be protonated.13 The pKa values deducedfrom the curve are 6.8 and 7.8, respectively. The lower pKa corresponds well to the second acid dissociation constantof phosphorous acid (the conjugate acid of phosphite), suggestingthat the enzyme may utilize the dianionic form of the substrate. Thehigher pKa would then correspond to anenzyme residue that must be protonated. One candidate would be His292(Figure 1), but if so, it could not be thecatalytic base that deprotonates the water nucleophile. One scenariothat would still allow His292 to serve as a base involves bindingof dianionic phosphite to PTDH with a protonated His292, followedby the transfer of a proton from His292 to phosphite in the ternarycomplex because of perturbed pKa valuesin the ternary complex (Figure 2). An alternativeinterpretation of the data is that phosphite must be protonated andHis292 deprotonated for substrate binding. In this interpretation,the minor, monoprotonated form of the substrate at the optimal pHof 7.25 would bind to the minor form of the enzyme with a deprotonatedHis (Figure 2). Such a reverse protonationscenario would result in an identical pH–rate profile becauseboth of these two protonation states of the substrate and enzyme wouldhave maximal occupancy at pH 7.25, decreasing at both higher and lowerpH values.13 Because the pKa values of the acidic and basic limbs of the PTDH pH–rateprofile are separated by <2 pH units, it is difficult to differentiatebetween normal and reverse protonation states.16

Bottom Line: Kinetic analysis of site-directed mutants of this residue shows that it is important for efficient catalysis, with an ~100-fold decrease in k(cat) and an almost 700-fold increase in K(m,phosphite) for the R301A mutant.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.All of the mutants had k(cat) values similar to that of the wild-type enzyme, indicating these residues are not important for catalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA.

ABSTRACT
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 k(cat) and an almost 700-fold increase in K(m,phosphite) for the R301A mutant. Interestingly, the R301K mutant displayed a slightly higher k(cat) than the parent PTDH, and a more modest increase in K(m) 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 k(cat) values similar to that of the wild-type enzyme, indicating these residues are not important for catalysis.

Show MeSH