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Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily.

Barrozo A, Duarte F, Bauer P, Carvalho AT, Kamerlin SC - J. Am. Chem. Soc. (2015)

Bottom Line: In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution.Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates.By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, BMC Box 596, SE-751 24, Uppsala, Sweden.

ABSTRACT
It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.

No MeSH data available.


Calculatedand experimentally derived activation energies for theenzyme-catalyzed reactions of the five substrates studied here bythe wild-type forms of (A) RlPMH and (B) BcPMH.61
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fig4: Calculatedand experimentally derived activation energies for theenzyme-catalyzed reactions of the five substrates studied here bythe wild-type forms of (A) RlPMH and (B) BcPMH.61

Mentions: As mentioned before,the reactions examined in this work correspondto the third step (III → IV) of thecatalytic cycle shown in Figure 3. Since experimentsshow that the hemiacetal cleavage is a fast step, we focused onlyon this second step, which is the most chemically challenging stepof the cycle, being thus the one related to the measured kinetic parameters.Figure 4 shows a comparison between our calculatedand, where available, experimental activation free energies (derivedfrom kcat, which provides an upper limitfor the reaction rate).8,12 The corresponding tabulated valuescan be found in Table 1. From our results,it can be seen that the model used in the present work reproducesthe experimental activation free energies within an accuracy of 1.7kcal·mol–1 for all substrates. It has additionallybeen argued8 that the PMHs considered inthis work can accept both diesters and phosphonates with such highproficiency in the same active site due to similar geometrical andsteric demands for the respective substrates and transition states.To probe this further, we have examined transition-state geometriesfor all uncatalyzed and enzyme-catalyzed reactions considered in thiswork. Table 2 shows a comparison of P(S)–Odistances to the oxygen atoms of the incoming nucleophile and departingleaving group for all substrates and reactions. Representative transition-statestructures in the BcPMH active site are also illustratedin Figure 5. From these results, it can beseen that the PMHs hydrolyze all substrates through a unified mechanismwith similar substrate binding positions and transition states. Withthe exception of the phosphonate, little change is seen in transition-stategeometry upon moving from aqueous solution to the enzyme active sites,in agreement with related experimental work by Herschlag and co-workers,22 as well as theoretical analysis by Hou and Cui,25 on alkaline phosphatase. Even in the case ofthe phosphonate, the overall transition-state size (considering thedistance between Onuc–Olg) stays verysimilar, and the main change is that the symmetry of the transitionstates changes, with P–Onuc becoming slightly elongatedand P–Olg slightly compressed compared to the correspondinguncatalyzed reaction. Hence, as suggested in previous works22,25 for alkaline phosphatase, we find very little effect on the transition-stategeometries of moving to the enzyme active when compared with thoseobtained through modeling the corresponding uncatalyzed reaction inaqueous solution.


Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily.

Barrozo A, Duarte F, Bauer P, Carvalho AT, Kamerlin SC - J. Am. Chem. Soc. (2015)

Calculatedand experimentally derived activation energies for theenzyme-catalyzed reactions of the five substrates studied here bythe wild-type forms of (A) RlPMH and (B) BcPMH.61
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4513756&req=5

fig4: Calculatedand experimentally derived activation energies for theenzyme-catalyzed reactions of the five substrates studied here bythe wild-type forms of (A) RlPMH and (B) BcPMH.61
Mentions: As mentioned before,the reactions examined in this work correspondto the third step (III → IV) of thecatalytic cycle shown in Figure 3. Since experimentsshow that the hemiacetal cleavage is a fast step, we focused onlyon this second step, which is the most chemically challenging stepof the cycle, being thus the one related to the measured kinetic parameters.Figure 4 shows a comparison between our calculatedand, where available, experimental activation free energies (derivedfrom kcat, which provides an upper limitfor the reaction rate).8,12 The corresponding tabulated valuescan be found in Table 1. From our results,it can be seen that the model used in the present work reproducesthe experimental activation free energies within an accuracy of 1.7kcal·mol–1 for all substrates. It has additionallybeen argued8 that the PMHs considered inthis work can accept both diesters and phosphonates with such highproficiency in the same active site due to similar geometrical andsteric demands for the respective substrates and transition states.To probe this further, we have examined transition-state geometriesfor all uncatalyzed and enzyme-catalyzed reactions considered in thiswork. Table 2 shows a comparison of P(S)–Odistances to the oxygen atoms of the incoming nucleophile and departingleaving group for all substrates and reactions. Representative transition-statestructures in the BcPMH active site are also illustratedin Figure 5. From these results, it can beseen that the PMHs hydrolyze all substrates through a unified mechanismwith similar substrate binding positions and transition states. Withthe exception of the phosphonate, little change is seen in transition-stategeometry upon moving from aqueous solution to the enzyme active sites,in agreement with related experimental work by Herschlag and co-workers,22 as well as theoretical analysis by Hou and Cui,25 on alkaline phosphatase. Even in the case ofthe phosphonate, the overall transition-state size (considering thedistance between Onuc–Olg) stays verysimilar, and the main change is that the symmetry of the transitionstates changes, with P–Onuc becoming slightly elongatedand P–Olg slightly compressed compared to the correspondinguncatalyzed reaction. Hence, as suggested in previous works22,25 for alkaline phosphatase, we find very little effect on the transition-stategeometries of moving to the enzyme active when compared with thoseobtained through modeling the corresponding uncatalyzed reaction inaqueous solution.

Bottom Line: In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution.Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates.By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, BMC Box 596, SE-751 24, Uppsala, Sweden.

ABSTRACT
It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.

No MeSH data available.