Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykAPA and PykFPA. In other well-characterized organisms cont...

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Autores principales:	Yassmin Abdelhamid, Meng Wang, Susannah L. Parkhill, Paul Brear, Xavier Chee, Taufiq Rahman, Martin Welch
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	bacterial metabolism Entner-Doudoroff pathway glycolysis Pseudomonas aeruginosa pyruvate kinase pykF Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/21e783ea8e5c41c5b71354e45a426e52
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spelling	oai:doaj.org-article:21e783ea8e5c41c5b71354e45a426e522021-11-16T10:42:37ZStructure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa1664-302X10.3389/fmicb.2021.790742https://doaj.org/article/21e783ea8e5c41c5b71354e45a426e522021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmicb.2021.790742/fullhttps://doaj.org/toc/1664-302XPseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykAPA and PykFPA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykAPA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykFPA isozyme. We show that pykFPA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykAPA and PykFPA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykFPA revealed significant differences compared with PykAPA. Notably, although the main allosteric regulator binding-site of PykFPA was empty, the “ring loop” covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent “locked on” and “locked off” allosteric activation phenotypes, depending on the residue mutated. Analysis of PykFPA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.Yassmin AbdelhamidMeng WangSusannah L. ParkhillPaul BrearXavier CheeTaufiq RahmanMartin WelchFrontiers Media S.A.articlebacterial metabolismEntner-Doudoroff pathwayglycolysisPseudomonas aeruginosapyruvate kinasepykFMicrobiologyQR1-502ENFrontiers in Microbiology, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	bacterial metabolism Entner-Doudoroff pathway glycolysis Pseudomonas aeruginosa pyruvate kinase pykF Microbiology QR1-502
spellingShingle	bacterial metabolism Entner-Doudoroff pathway glycolysis Pseudomonas aeruginosa pyruvate kinase pykF Microbiology QR1-502 Yassmin Abdelhamid Meng Wang Susannah L. Parkhill Paul Brear Xavier Chee Taufiq Rahman Martin Welch Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa
description	Pseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykAPA and PykFPA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykAPA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykFPA isozyme. We show that pykFPA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykAPA and PykFPA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykFPA revealed significant differences compared with PykAPA. Notably, although the main allosteric regulator binding-site of PykFPA was empty, the “ring loop” covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent “locked on” and “locked off” allosteric activation phenotypes, depending on the residue mutated. Analysis of PykFPA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.
format	article
author	Yassmin Abdelhamid Meng Wang Susannah L. Parkhill Paul Brear Xavier Chee Taufiq Rahman Martin Welch
author_facet	Yassmin Abdelhamid Meng Wang Susannah L. Parkhill Paul Brear Xavier Chee Taufiq Rahman Martin Welch
author_sort	Yassmin Abdelhamid
title	Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa
title_short	Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa
title_full	Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa
title_fullStr	Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa
title_full_unstemmed	Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa
title_sort	structure, function and regulation of a second pyruvate kinase isozyme in pseudomonas aeruginosa
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/21e783ea8e5c41c5b71354e45a426e52
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_version_	1718426531002318848

Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa

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