Arg126 and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not
Prostaglandins are signaling molecules that regulate different physiological processes, involving allergic and inflammatory responses and cardiovascular control. They are involved in several pathophysiological processes, including inflammation and cancer. The inducible terminal enzyme, microsomal pr...
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description | Prostaglandins are signaling molecules that regulate different physiological processes, involving allergic and inflammatory responses and cardiovascular control. They are involved in several pathophysiological processes, including inflammation and cancer. The inducible terminal enzyme, microsomal prostaglandin E synthase 1 (MPGES1), catalyses prostaglandin E2 production during inflammation. MPGES1 has therefore been intensively studied as a pharmaceutical target and many competitive inhibitors targeting its active site have been developed. However, little is known about its catalytic mechanism.
The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1.
Based on results and predictions from previous structural studies, the amino acid residues Asp49, Arg73, Arg126, and Ser127 were chosen and altered by site-directed mutagenesis. The mutated enzyme variants were cloned and expressed in both the E. coli and the Baculovirus expression systems. Their catalytic significance was evaluated by activity measurements with prostanoid profiling.
Our study shows that Arg126 and Asp49 are absolutely required for the catalytic activity of MPGES1, as when exchanged, the enzyme variants loose activity. Ser127 and Arg73 on the other hand, don't seem to be central to the catalytic mechanism because when exchanged, their variants retain considerable activity. Our finding that the Ser127Ala variant retains activity was surprising since high-resolution structural data supported a role in glutathione activation. The close proximity of Ser127 to the active site is, however, supported since the Ser127Cys variant displays 80% lowered activity. |
doi_str_mv | 10.1371/journal.pone.0163600 |
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The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1.
Based on results and predictions from previous structural studies, the amino acid residues Asp49, Arg73, Arg126, and Ser127 were chosen and altered by site-directed mutagenesis. The mutated enzyme variants were cloned and expressed in both the E. coli and the Baculovirus expression systems. Their catalytic significance was evaluated by activity measurements with prostanoid profiling.
Our study shows that Arg126 and Asp49 are absolutely required for the catalytic activity of MPGES1, as when exchanged, the enzyme variants loose activity. Ser127 and Arg73 on the other hand, don't seem to be central to the catalytic mechanism because when exchanged, their variants retain considerable activity. Our finding that the Ser127Ala variant retains activity was surprising since high-resolution structural data supported a role in glutathione activation. The close proximity of Ser127 to the active site is, however, supported since the Ser127Cys variant displays 80% lowered activity.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0163600</identifier><identifier>PMID: 27684486</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Amino acids ; Baculoviridae ; Baculovirus ; Biochemistry ; Biology and Life Sciences ; Cancer ; Cardiovascular system ; Catalysis ; Catalytic activity ; E coli ; Environmental health ; Enzymes ; Escherichia coli ; Glutathione ; Hypersensitivity ; Inflammation ; Medicin och hälsovetenskap ; Medicine ; Medicine and Health Sciences ; Morgenstern, Linda ; Mutagenesis ; Physical Sciences ; Physiology ; Prostaglandin E ; Prostaglandin E2 ; Prostaglandins ; Research and Analysis Methods ; Rheumatology ; Rodents ; Signaling ; Site-directed mutagenesis ; Toxicology</subject><ispartof>PloS one, 2016-09, Vol.11 (9), p.e0163600</ispartof><rights>This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication: https://creativecommons.org/publicdomain/zero/1.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c609t-e972d35c8795fe87cd1fb5155fc63f2659e16a1401f33b642392ec0684162aa3</citedby><cites>FETCH-LOGICAL-c609t-e972d35c8795fe87cd1fb5155fc63f2659e16a1401f33b642392ec0684162aa3</cites><orcidid>0000-0001-6808-7024</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042469/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042469/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,552,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27684486$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttp://kipublications.ki.se/Default.aspx?queryparsed=id:134429611$$DView record from Swedish Publication Index$$Hfree_for_read</backlink></links><search><contributor>Parker, Emily J.</contributor><creatorcontrib>Raouf, Joan</creatorcontrib><creatorcontrib>Rafique, Nazmi</creatorcontrib><creatorcontrib>Goodman, Michael Christopher</creatorcontrib><creatorcontrib>Idborg, Helena</creatorcontrib><creatorcontrib>Bergqvist, Filip</creatorcontrib><creatorcontrib>Armstrong, Richard N</creatorcontrib><creatorcontrib>Jakobsson, Per-Johan</creatorcontrib><creatorcontrib>Morgenstern, Ralf</creatorcontrib><creatorcontrib>Spahiu, Linda</creatorcontrib><title>Arg126 and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Prostaglandins are signaling molecules that regulate different physiological processes, involving allergic and inflammatory responses and cardiovascular control. They are involved in several pathophysiological processes, including inflammation and cancer. The inducible terminal enzyme, microsomal prostaglandin E synthase 1 (MPGES1), catalyses prostaglandin E2 production during inflammation. MPGES1 has therefore been intensively studied as a pharmaceutical target and many competitive inhibitors targeting its active site have been developed. However, little is known about its catalytic mechanism.
The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1.
Based on results and predictions from previous structural studies, the amino acid residues Asp49, Arg73, Arg126, and Ser127 were chosen and altered by site-directed mutagenesis. The mutated enzyme variants were cloned and expressed in both the E. coli and the Baculovirus expression systems. Their catalytic significance was evaluated by activity measurements with prostanoid profiling.
Our study shows that Arg126 and Asp49 are absolutely required for the catalytic activity of MPGES1, as when exchanged, the enzyme variants loose activity. Ser127 and Arg73 on the other hand, don't seem to be central to the catalytic mechanism because when exchanged, their variants retain considerable activity. Our finding that the Ser127Ala variant retains activity was surprising since high-resolution structural data supported a role in glutathione activation. The close proximity of Ser127 to the active site is, however, supported since the Ser127Cys variant displays 80% lowered activity.</description><subject>Amino acids</subject><subject>Baculoviridae</subject><subject>Baculovirus</subject><subject>Biochemistry</subject><subject>Biology and Life Sciences</subject><subject>Cancer</subject><subject>Cardiovascular system</subject><subject>Catalysis</subject><subject>Catalytic activity</subject><subject>E coli</subject><subject>Environmental health</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Glutathione</subject><subject>Hypersensitivity</subject><subject>Inflammation</subject><subject>Medicin och hälsovetenskap</subject><subject>Medicine</subject><subject>Medicine and Health Sciences</subject><subject>Morgenstern, Linda</subject><subject>Mutagenesis</subject><subject>Physical Sciences</subject><subject>Physiology</subject><subject>Prostaglandin E</subject><subject>Prostaglandin E2</subject><subject>Prostaglandins</subject><subject>Research and Analysis Methods</subject><subject>Rheumatology</subject><subject>Rodents</subject><subject>Signaling</subject><subject>Site-directed 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and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not</title><author>Raouf, Joan ; Rafique, Nazmi ; Goodman, Michael Christopher ; Idborg, Helena ; Bergqvist, Filip ; Armstrong, Richard N ; Jakobsson, Per-Johan ; Morgenstern, Ralf ; Spahiu, Linda</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c609t-e972d35c8795fe87cd1fb5155fc63f2659e16a1401f33b642392ec0684162aa3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Amino acids</topic><topic>Baculoviridae</topic><topic>Baculovirus</topic><topic>Biochemistry</topic><topic>Biology and Life Sciences</topic><topic>Cancer</topic><topic>Cardiovascular system</topic><topic>Catalysis</topic><topic>Catalytic activity</topic><topic>E coli</topic><topic>Environmental health</topic><topic>Enzymes</topic><topic>Escherichia coli</topic><topic>Glutathione</topic><topic>Hypersensitivity</topic><topic>Inflammation</topic><topic>Medicin och hälsovetenskap</topic><topic>Medicine</topic><topic>Medicine and Health Sciences</topic><topic>Morgenstern, Linda</topic><topic>Mutagenesis</topic><topic>Physical Sciences</topic><topic>Physiology</topic><topic>Prostaglandin E</topic><topic>Prostaglandin E2</topic><topic>Prostaglandins</topic><topic>Research and Analysis Methods</topic><topic>Rheumatology</topic><topic>Rodents</topic><topic>Signaling</topic><topic>Site-directed mutagenesis</topic><topic>Toxicology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Raouf, Joan</creatorcontrib><creatorcontrib>Rafique, Nazmi</creatorcontrib><creatorcontrib>Goodman, Michael Christopher</creatorcontrib><creatorcontrib>Idborg, Helena</creatorcontrib><creatorcontrib>Bergqvist, Filip</creatorcontrib><creatorcontrib>Armstrong, Richard N</creatorcontrib><creatorcontrib>Jakobsson, 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J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Arg126 and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2016-09-01</date><risdate>2016</risdate><volume>11</volume><issue>9</issue><spage>e0163600</spage><pages>e0163600-</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Prostaglandins are signaling molecules that regulate different physiological processes, involving allergic and inflammatory responses and cardiovascular control. They are involved in several pathophysiological processes, including inflammation and cancer. The inducible terminal enzyme, microsomal prostaglandin E synthase 1 (MPGES1), catalyses prostaglandin E2 production during inflammation. MPGES1 has therefore been intensively studied as a pharmaceutical target and many competitive inhibitors targeting its active site have been developed. However, little is known about its catalytic mechanism.
The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1.
Based on results and predictions from previous structural studies, the amino acid residues Asp49, Arg73, Arg126, and Ser127 were chosen and altered by site-directed mutagenesis. The mutated enzyme variants were cloned and expressed in both the E. coli and the Baculovirus expression systems. Their catalytic significance was evaluated by activity measurements with prostanoid profiling.
Our study shows that Arg126 and Asp49 are absolutely required for the catalytic activity of MPGES1, as when exchanged, the enzyme variants loose activity. Ser127 and Arg73 on the other hand, don't seem to be central to the catalytic mechanism because when exchanged, their variants retain considerable activity. Our finding that the Ser127Ala variant retains activity was surprising since high-resolution structural data supported a role in glutathione activation. The close proximity of Ser127 to the active site is, however, supported since the Ser127Cys variant displays 80% lowered activity.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>27684486</pmid><doi>10.1371/journal.pone.0163600</doi><orcidid>https://orcid.org/0000-0001-6808-7024</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | Amino acids Baculoviridae Baculovirus Biochemistry Biology and Life Sciences Cancer Cardiovascular system Catalysis Catalytic activity E coli Environmental health Enzymes Escherichia coli Glutathione Hypersensitivity Inflammation Medicin och hälsovetenskap Medicine Medicine and Health Sciences Morgenstern, Linda Mutagenesis Physical Sciences Physiology Prostaglandin E Prostaglandin E2 Prostaglandins Research and Analysis Methods Rheumatology Rodents Signaling Site-directed mutagenesis Toxicology |
title | Arg126 and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not |
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