Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis
In an effort to probe the structure, mechanism, and biochemical properties of metallo-β-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies s...
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| Veröffentlicht in: | Journal of the American Chemical Society 2009-08, Vol.131 (30), p.10753-10762 |
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| container_title | Journal of the American Chemical Society |
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| creator | Hawk, Megan J Breece, Robert M Hajdin, Christine E Bender, Katherine M Hu, Zhenxin Costello, Alison L Bennett, Brian Tierney, David L Crowder, Michael W |
| description | In an effort to probe the structure, mechanism, and biochemical properties of metallo-β-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV−vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms. |
| doi_str_mv | 10.1021/ja900296u |
| format | Article |
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Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV−vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.</description><identifier>ISSN: 0002-7863</identifier><identifier>EISSN: 1520-5126</identifier><identifier>DOI: 10.1021/ja900296u</identifier><identifier>PMID: 19588962</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>BACILLUS ; Bacillus anthracis - enzymology ; BASIC BIOLOGICAL SCIENCES ; beta-Lactamases - biosynthesis ; beta-Lactamases - metabolism ; Cobalt - metabolism ; ELECTRON SPIN RESONANCE ; ENZYMES ; GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE ; KINETICS ; METALS ; national synchrotron light source ; NUCLEAR MAGNETIC RESONANCE ; Protein Binding ; PROTEINS ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - metabolism ; Spectrum Analysis ; Substrate Specificity ; Zinc - metabolism</subject><ispartof>Journal of the American Chemical Society, 2009-08, Vol.131 (30), p.10753-10762</ispartof><rights>Copyright © 2009 American Chemical Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a430t-fe4630bd193bd573010b83963cea73fb7a04b7ff31d372fe1dd36d6a2c524da23</citedby><cites>FETCH-LOGICAL-a430t-fe4630bd193bd573010b83963cea73fb7a04b7ff31d372fe1dd36d6a2c524da23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/ja900296u$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/ja900296u$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>230,314,780,784,885,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19588962$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/980146$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Hawk, Megan J</creatorcontrib><creatorcontrib>Breece, Robert M</creatorcontrib><creatorcontrib>Hajdin, Christine E</creatorcontrib><creatorcontrib>Bender, Katherine M</creatorcontrib><creatorcontrib>Hu, Zhenxin</creatorcontrib><creatorcontrib>Costello, Alison L</creatorcontrib><creatorcontrib>Bennett, Brian</creatorcontrib><creatorcontrib>Tierney, David L</creatorcontrib><creatorcontrib>Crowder, Michael W</creatorcontrib><creatorcontrib>Brookhaven National Laboratory (BNL) National Synchrotron Light Source</creatorcontrib><title>Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis</title><title>Journal of the American Chemical Society</title><addtitle>J. Am. Chem. Soc</addtitle><description>In an effort to probe the structure, mechanism, and biochemical properties of metallo-β-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV−vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.</description><subject>BACILLUS</subject><subject>Bacillus anthracis - enzymology</subject><subject>BASIC BIOLOGICAL SCIENCES</subject><subject>beta-Lactamases - biosynthesis</subject><subject>beta-Lactamases - metabolism</subject><subject>Cobalt - metabolism</subject><subject>ELECTRON SPIN RESONANCE</subject><subject>ENZYMES</subject><subject>GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE</subject><subject>KINETICS</subject><subject>METALS</subject><subject>national synchrotron light source</subject><subject>NUCLEAR MAGNETIC RESONANCE</subject><subject>Protein Binding</subject><subject>PROTEINS</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - metabolism</subject><subject>Spectrum Analysis</subject><subject>Substrate Specificity</subject><subject>Zinc - metabolism</subject><issn>0002-7863</issn><issn>1520-5126</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkctuEzEUhi0EoiGw4AWQWaDSxYAvc90gkVAgUhAb2LDAOuNL48hjF9tTidfiQfpMmCYqRWJlH5_Pv__jH6GnlLyihNHXexgIYUM730ML2jBSNZS199GClNOq61t-gh6ltC9lzXr6EJ3Qoen7oWUL9P2dNUZH7bMFh1fWK-svcDB4HV5uNmcYvMLf_M02B_xJZ3AuVNe_qi3IDBMkjVcOGDYxTHgF0jo3p3Ir72Ip0mP0wIBL-slxXaKv78-_rD9W288fNuu32wpqTnJldN1yMio68FE1HSeUjD0fWi41dNyMHZB67IzhVPGOGU2V4q1qgcmG1QoYX6I3B93LeZy0kmWeCE5cRjtB_CkCWPFvx9uduAhXgrOhabquCDw_CISUrUjSZi13MnivZRZDT2gxuESnx0di-DHrlMVkk9TOgddhTqLjNeF13deFPDuQMoaUoja3TigRfyITt5EV9tld63_JY0YFeHEAQCaxD3P05Sf_I_QbpaydKg</recordid><startdate>20090805</startdate><enddate>20090805</enddate><creator>Hawk, Megan J</creator><creator>Breece, Robert M</creator><creator>Hajdin, Christine E</creator><creator>Bender, Katherine M</creator><creator>Hu, Zhenxin</creator><creator>Costello, Alison L</creator><creator>Bennett, Brian</creator><creator>Tierney, David L</creator><creator>Crowder, Michael W</creator><general>American Chemical Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>OTOTI</scope><scope>5PM</scope></search><sort><creationdate>20090805</creationdate><title>Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis</title><author>Hawk, Megan J ; Breece, Robert M ; Hajdin, Christine E ; Bender, Katherine M ; Hu, Zhenxin ; Costello, Alison L ; Bennett, Brian ; Tierney, David L ; Crowder, Michael W</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a430t-fe4630bd193bd573010b83963cea73fb7a04b7ff31d372fe1dd36d6a2c524da23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>BACILLUS</topic><topic>Bacillus anthracis - enzymology</topic><topic>BASIC BIOLOGICAL SCIENCES</topic><topic>beta-Lactamases - biosynthesis</topic><topic>beta-Lactamases - metabolism</topic><topic>Cobalt - metabolism</topic><topic>ELECTRON SPIN RESONANCE</topic><topic>ENZYMES</topic><topic>GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE</topic><topic>KINETICS</topic><topic>METALS</topic><topic>national synchrotron light source</topic><topic>NUCLEAR MAGNETIC RESONANCE</topic><topic>Protein Binding</topic><topic>PROTEINS</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - metabolism</topic><topic>Spectrum Analysis</topic><topic>Substrate Specificity</topic><topic>Zinc - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hawk, Megan J</creatorcontrib><creatorcontrib>Breece, Robert M</creatorcontrib><creatorcontrib>Hajdin, Christine E</creatorcontrib><creatorcontrib>Bender, Katherine M</creatorcontrib><creatorcontrib>Hu, Zhenxin</creatorcontrib><creatorcontrib>Costello, Alison L</creatorcontrib><creatorcontrib>Bennett, Brian</creatorcontrib><creatorcontrib>Tierney, David L</creatorcontrib><creatorcontrib>Crowder, Michael W</creatorcontrib><creatorcontrib>Brookhaven National Laboratory (BNL) National Synchrotron Light Source</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>OSTI.GOV</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of the American Chemical Society</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hawk, Megan J</au><au>Breece, Robert M</au><au>Hajdin, Christine E</au><au>Bender, Katherine M</au><au>Hu, Zhenxin</au><au>Costello, Alison L</au><au>Bennett, Brian</au><au>Tierney, David L</au><au>Crowder, Michael W</au><aucorp>Brookhaven National Laboratory (BNL) National Synchrotron Light Source</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis</atitle><jtitle>Journal of the American Chemical Society</jtitle><addtitle>J. Am. Chem. Soc</addtitle><date>2009-08-05</date><risdate>2009</risdate><volume>131</volume><issue>30</issue><spage>10753</spage><epage>10762</epage><pages>10753-10762</pages><issn>0002-7863</issn><eissn>1520-5126</eissn><abstract>In an effort to probe the structure, mechanism, and biochemical properties of metallo-β-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV−vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>19588962</pmid><doi>10.1021/ja900296u</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; American Chemical Society Journals |
| subjects | BACILLUS Bacillus anthracis - enzymology BASIC BIOLOGICAL SCIENCES beta-Lactamases - biosynthesis beta-Lactamases - metabolism Cobalt - metabolism ELECTRON SPIN RESONANCE ENZYMES GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE KINETICS METALS national synchrotron light source NUCLEAR MAGNETIC RESONANCE Protein Binding PROTEINS Recombinant Proteins - biosynthesis Recombinant Proteins - metabolism Spectrum Analysis Substrate Specificity Zinc - metabolism |
| title | Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis |
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