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Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2015 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, no 28, 9061-9076 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2015. Vol. 137, no 28, 9061-9076 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-260856DOI: 10.1021/jacs.5b03945ISI: 000358556200033PubMedID: 26091851OAI: oai:DiVA.org:uu-260856DiVA: diva2:848807
Funder
Swedish Research Council, 2010-5026EU, FP7, Seventh Framework Programme, 306474Swedish National Infrastructure for Computing (SNIC), 25/2-10
Note

De 2 första författarna delar förstaförfattarskapet.

Available from: 2015-08-26 Created: 2015-08-25 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Promiscuity and Selectivity in Phosphoryl Transferases
Open this publication in new window or tab >>Promiscuity and Selectivity in Phosphoryl Transferases
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Phosphoryl transfers are essential chemical reactions in key life processes, including energy production, signal transduction and protein synthesis. They are known for having extremely low reaction rates in aqueous solution, reaching the scale of millions of years. In order to make life possible, enzymes that catalyse phosphoryl transfer, phosphoryl transferases, have evolved to be tremendously proficient catalysts, increasing reaction rates to the millisecond timescale.

Due to the nature of the electronic structure of phosphorus atoms, understanding how hydrolysis of phosphate esters occurs is a complex task. Experimental studies on the hydrolysis of phosphate monoesters with acidic leaving groups suggest a concerted mechanism with a loose, metaphosphate-like transition state. Theoretical studies have suggested two possible concerted pathways, either with loose or tight transition state geometries, plus the possibility of a stepwise mechanism with the formation of a phosphorane intermediate. Different pathways were shown to be energetically preferable depending on the acidity of the leaving group. Here we performed computational studies to revisit how this mechanistic shift occurs along a series of aryl phosphate monoesters, suggesting possible factors leading to such change.

The fact that distinct pathways can occur in solution could mean that the same is possible for an enzyme active site. We performed simulations on the catalytic activity of β-phosphoglucomutase, suggesting that it is possible for two mechanisms to occur at the same time for the phosphoryl transfer.

Curiously, several phosphoryl transferases were shown to be able to catalyse not only phosphate ester hydrolysis, but also the cleavage of other compounds. We modeled the catalytic mechanism of two highly promiscuous members of the alkaline phosphatase superfamily. Our model reproduces key experimental observables and shows that these enzymes are electrostatically flexible, employing the same set of residues to enhance the rates of different reactions, with different electrostatic contributions per residue.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 74 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1350
Keyword
phosphate chemistry, linear free energy relationships, phosphatase, catalytic promiscuity, empirical valence bond approach, alkaline phosphatase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-279693 (URN)978-91-554-9497-1 (ISBN)
Public defence
2016-04-25, C8:305, BMC, Husarg. 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2016-04-04 Created: 2016-03-03 Last updated: 2016-04-12

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Barrozo, AlexandreDuarte, FernandaBauer, PaulKamerlin, Shina C. Lynn

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