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Promiscuity and Selectivity in Phosphoryl Transferases
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
2016 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2016. , s. 74
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1350
Emneord [en]
phosphate chemistry, linear free energy relationships, phosphatase, catalytic promiscuity, empirical valence bond approach, alkaline phosphatase
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-279693ISBN: 978-91-554-9497-1 (tryckt)OAI: oai:DiVA.org:uu-279693DiVA, id: diva2:908672
Disputas
2016-04-25, C8:305, BMC, Husarg. 3, Uppsala, 13:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2016-04-04 Laget: 2016-03-03 Sist oppdatert: 2016-04-12
Delarbeid
1. Evaluation and Characterisation of Mechanistic Alternatives for beta-Phosphoglucomutase
Åpne denne publikasjonen i ny fane eller vindu >>Evaluation and Characterisation of Mechanistic Alternatives for beta-Phosphoglucomutase
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-278943 (URN)
Tilgjengelig fra: 2016-02-26 Laget: 2016-02-26 Sist oppdatert: 2016-04-12
2. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
Åpne denne publikasjonen i ny fane eller vindu >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
Vise andre…
2014 (engelsk)Inngår i: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, nr 16, s. 4351-4362Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
Forskningsfinansiär
Swedish National Infrastructure for Computing (SNIC), 2013/26-1
Tilgjengelig fra: 2014-06-23 Laget: 2014-06-04 Sist oppdatert: 2018-12-03bibliografisk kontrollert
3. Mechanistic Shifts Along the Linear Free Energy Relationship for Aryl Phosphate Monoester Hydrolysis
Åpne denne publikasjonen i ny fane eller vindu >>Mechanistic Shifts Along the Linear Free Energy Relationship for Aryl Phosphate Monoester Hydrolysis
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-278945 (URN)
Tilgjengelig fra: 2016-02-26 Laget: 2016-02-26 Sist oppdatert: 2016-04-12
4. Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily
Åpne denne publikasjonen i ny fane eller vindu >>Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily
Vise andre…
2015 (engelsk)Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, nr 28, s. 9061-9076Artikkel i tidsskrift (Fagfellevurdert) 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.

HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-260856 (URN)10.1021/jacs.5b03945 (DOI)000358556200033 ()26091851 (PubMedID)
Forskningsfinansiär
Swedish Research Council, 2010-5026EU, FP7, Seventh Framework Programme, 306474Swedish National Infrastructure for Computing (SNIC), 25/2-10
Merknad

De 2 första författarna delar förstaförfattarskapet.

Tilgjengelig fra: 2015-08-26 Laget: 2015-08-25 Sist oppdatert: 2017-12-04bibliografisk kontrollert

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