5aj9: Difference between revisions
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The | ==G7 mutant of PAS, arylsulfatase from Pseudomonas Aeruginosa== | ||
<StructureSection load='5aj9' size='340' side='right'caption='[[5aj9]], [[Resolution|resolution]] 2.00Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[5aj9]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Pseudomonas_aeruginosa Pseudomonas aeruginosa]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5AJ9 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5AJ9 FirstGlance]. <br> | |||
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=DDZ:3,3-DIHYDROXY+L-ALANINE'>DDZ</scene>, <scene name='pdbligand=MES:2-(N-MORPHOLINO)-ETHANESULFONIC+ACID'>MES</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=5aj9 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5aj9 OCA], [https://pdbe.org/5aj9 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5aj9 RCSB], [https://www.ebi.ac.uk/pdbsum/5aj9 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5aj9 ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/ARS_PSEAE ARS_PSEAE] | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (</=50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E*S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (betaleavinggroup from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes. | |||
Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset.,Miton CM, Jonas S, Fischer G, Duarte F, Mohamed MF, van Loo B, Kintses B, Kamerlin SCL, Tokuriki N, Hyvonen M, Hollfelder F Proc Natl Acad Sci U S A. 2018 Jul 31;115(31):E7293-E7302. doi:, 10.1073/pnas.1607817115. Epub 2018 Jul 16. PMID:30012610<ref>PMID:30012610</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
[[Category: | <div class="pdbe-citations 5aj9" style="background-color:#fffaf0;"></div> | ||
[[Category: | |||
[[Category: Hollfelder | ==See Also== | ||
[[Category: | *[[Sulfatase 3D structures|Sulfatase 3D structures]] | ||
[[Category: | == References == | ||
[[Category: | <references/> | ||
[[Category: | __TOC__ | ||
[[Category: | </StructureSection> | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: Pseudomonas aeruginosa]] | |||
[[Category: Fischer G]] | |||
[[Category: Hollfelder F]] | |||
[[Category: Hyvonen M]] | |||
[[Category: Jonas S]] | |||
[[Category: Kintses B]] | |||
[[Category: Loo Bv]] | |||
[[Category: Miton CM]] | |||
[[Category: Mohammed MF]] | |||
[[Category: Tokuriki N]] | |||
Latest revision as of 14:37, 6 November 2024
G7 mutant of PAS, arylsulfatase from Pseudomonas AeruginosaG7 mutant of PAS, arylsulfatase from Pseudomonas Aeruginosa
Structural highlights
FunctionPublication Abstract from PubMedThe recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (</=50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E*S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (betaleavinggroup from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes. Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset.,Miton CM, Jonas S, Fischer G, Duarte F, Mohamed MF, van Loo B, Kintses B, Kamerlin SCL, Tokuriki N, Hyvonen M, Hollfelder F Proc Natl Acad Sci U S A. 2018 Jul 31;115(31):E7293-E7302. doi:, 10.1073/pnas.1607817115. Epub 2018 Jul 16. PMID:30012610[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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