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Purification and characterization of the soluble methane monooxygenase of the type II methanotrophic bacterium Methylocystis sp. strain WI 14
Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with th...
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| Published in: | Applied and environmental microbiology 1999-09, Vol.65 (9), p.3929-3935 |
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| container_end_page | 3935 |
| container_issue | 9 |
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| container_title | Applied and environmental microbiology |
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| creator | GROSSE, S LARAMEE, L WENDLANDT, K.-D MCDONALD, I. R MIGUEZ, C. B KLEBER, H.-P |
| description | Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation ( |
| doi_str_mv | 10.1128/aem.65.9.3929-3935.1999 |
| format | article |
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R ; MIGUEZ, C. B ; KLEBER, H.-P</creator><creatorcontrib>GROSSE, S ; LARAMEE, L ; WENDLANDT, K.-D ; MCDONALD, I. R ; MIGUEZ, C. B ; KLEBER, H.-P</creatorcontrib><description>Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 microM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an alpha(2)beta(2)gamma(2) structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg(2+) ions (with a total loss of enzyme activity at 0.01 mM Hg(2+)) and Cu(2+), Zn(2+), and Ni(2+) ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).</description><identifier>ISSN: 0099-2240</identifier><identifier>EISSN: 1098-5336</identifier><identifier>DOI: 10.1128/aem.65.9.3929-3935.1999</identifier><identifier>PMID: 10473397</identifier><identifier>CODEN: AEMIDF</identifier><language>eng</language><publisher>Washington, DC: American Society for Microbiology</publisher><subject>Alphaproteobacteria - enzymology ; Alphaproteobacteria - growth & development ; Bacteria ; Biological and medical sciences ; Biology of microorganisms of confirmed or potential industrial interest ; Biotechnology ; Cloning, Molecular ; DNA, Bacterial - genetics ; Enzyme Stability ; Enzymology and Protein Engineering ; Fundamental and applied biological sciences. Psychology ; Genes, Bacterial ; Methane ; Methylococcus capsulatus ; Methylocystis ; Methylosinus trichosporium ; Microbiology ; Miscellaneous ; Mission oriented research ; Oxygenases - genetics ; Oxygenases - isolation & purification ; Oxygenases - metabolism ; Phylogeny ; Polymerase Chain Reaction - methods ; RNA, Ribosomal, 16S - genetics ; Sequence Analysis, DNA ; Solubility ; Substrate Specificity</subject><ispartof>Applied and environmental microbiology, 1999-09, Vol.65 (9), p.3929-3935</ispartof><rights>1999 INIST-CNRS</rights><rights>Copyright American Society for Microbiology Sep 1999</rights><rights>Copyright © 1999, American Society for Microbiology 1999</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c498t-9fa8f862d59e0487c5e13109e3534b1097a6862363be6c68042cf40192ca06d63</citedby><cites>FETCH-LOGICAL-c498t-9fa8f862d59e0487c5e13109e3534b1097a6862363be6c68042cf40192ca06d63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC99722/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC99722/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,3188,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1958764$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10473397$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>GROSSE, S</creatorcontrib><creatorcontrib>LARAMEE, L</creatorcontrib><creatorcontrib>WENDLANDT, K.-D</creatorcontrib><creatorcontrib>MCDONALD, I. R</creatorcontrib><creatorcontrib>MIGUEZ, C. B</creatorcontrib><creatorcontrib>KLEBER, H.-P</creatorcontrib><title>Purification and characterization of the soluble methane monooxygenase of the type II methanotrophic bacterium Methylocystis sp. strain WI 14</title><title>Applied and environmental microbiology</title><addtitle>Appl Environ Microbiol</addtitle><description>Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 microM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an alpha(2)beta(2)gamma(2) structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg(2+) ions (with a total loss of enzyme activity at 0.01 mM Hg(2+)) and Cu(2+), Zn(2+), and Ni(2+) ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).</description><subject>Alphaproteobacteria - enzymology</subject><subject>Alphaproteobacteria - growth & development</subject><subject>Bacteria</subject><subject>Biological and medical sciences</subject><subject>Biology of microorganisms of confirmed or potential industrial interest</subject><subject>Biotechnology</subject><subject>Cloning, Molecular</subject><subject>DNA, Bacterial - genetics</subject><subject>Enzyme Stability</subject><subject>Enzymology and Protein Engineering</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genes, Bacterial</subject><subject>Methane</subject><subject>Methylococcus capsulatus</subject><subject>Methylocystis</subject><subject>Methylosinus trichosporium</subject><subject>Microbiology</subject><subject>Miscellaneous</subject><subject>Mission oriented research</subject><subject>Oxygenases - genetics</subject><subject>Oxygenases - isolation & purification</subject><subject>Oxygenases - metabolism</subject><subject>Phylogeny</subject><subject>Polymerase Chain Reaction - methods</subject><subject>RNA, Ribosomal, 16S - genetics</subject><subject>Sequence Analysis, DNA</subject><subject>Solubility</subject><subject>Substrate Specificity</subject><issn>0099-2240</issn><issn>1098-5336</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><recordid>eNqFksFu1DAQhi0EokvhFcBCiFuCHTuOR-JSVS2s1AoOII6W43UaV4m92AkivEPfGa-yQOGCfBhr5vtHM6MfoReUlJRW8o22YynqEkoGFRQMWF1SAHiANpSALGrGxEO0IQSgqCpOTtCTlG4JIZwI-RidUMIbxqDZoLuPc3SdM3pywWPtd9j0Omoz2eh-rMnQ4am3OIVhbgeLRzv12ucYfAjflxvrdbK_oGnZW7zdHqEwxbDvncHt2nAe8XUuLEMwS5pcwmlf4jRF7Tz-ssWUP0WPOj0k--wYT9Hny4tP5--Lqw_vtudnV4XhIKcCOi07KapdDZZw2ZjaUpYXt6xmvM2fRotcZoK1VhghCa9MxwmFymgidoKdordr3_3cjnZnrM9DDGof3ajjooJ26u-Kd726Cd8UQFNVWf76KI_h62zTpEaXjB2GfJcwJ9UQQjNH_gvShnHBKGTw5T_gbZijzzdQFamBy_wy1KyQiSGlaLvfA1OiDrZQZxfXStQK1MEW6mALdbBFVj6_v-893eqDDLw6AjoZPXRRe-PSHw5q2QjOfgKbdMNe</recordid><startdate>19990901</startdate><enddate>19990901</enddate><creator>GROSSE, S</creator><creator>LARAMEE, L</creator><creator>WENDLANDT, K.-D</creator><creator>MCDONALD, I. 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Psychology</topic><topic>Genes, Bacterial</topic><topic>Methane</topic><topic>Methylococcus capsulatus</topic><topic>Methylocystis</topic><topic>Methylosinus trichosporium</topic><topic>Microbiology</topic><topic>Miscellaneous</topic><topic>Mission oriented research</topic><topic>Oxygenases - genetics</topic><topic>Oxygenases - isolation & purification</topic><topic>Oxygenases - metabolism</topic><topic>Phylogeny</topic><topic>Polymerase Chain Reaction - methods</topic><topic>RNA, Ribosomal, 16S - genetics</topic><topic>Sequence Analysis, DNA</topic><topic>Solubility</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>GROSSE, S</creatorcontrib><creatorcontrib>LARAMEE, L</creatorcontrib><creatorcontrib>WENDLANDT, K.-D</creatorcontrib><creatorcontrib>MCDONALD, I. R</creatorcontrib><creatorcontrib>MIGUEZ, C. B</creatorcontrib><creatorcontrib>KLEBER, H.-P</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Environment Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Environment Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Applied and environmental microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>GROSSE, S</au><au>LARAMEE, L</au><au>WENDLANDT, K.-D</au><au>MCDONALD, I. R</au><au>MIGUEZ, C. B</au><au>KLEBER, H.-P</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and characterization of the soluble methane monooxygenase of the type II methanotrophic bacterium Methylocystis sp. strain WI 14</atitle><jtitle>Applied and environmental microbiology</jtitle><addtitle>Appl Environ Microbiol</addtitle><date>1999-09-01</date><risdate>1999</risdate><volume>65</volume><issue>9</issue><spage>3929</spage><epage>3935</epage><pages>3929-3935</pages><issn>0099-2240</issn><eissn>1098-5336</eissn><coden>AEMIDF</coden><abstract>Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 microM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an alpha(2)beta(2)gamma(2) structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg(2+) ions (with a total loss of enzyme activity at 0.01 mM Hg(2+)) and Cu(2+), Zn(2+), and Ni(2+) ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).</abstract><cop>Washington, DC</cop><pub>American Society for Microbiology</pub><pmid>10473397</pmid><doi>10.1128/aem.65.9.3929-3935.1999</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| source | American Society for Microbiology Journals; PubMed Central |
| subjects | Alphaproteobacteria - enzymology Alphaproteobacteria - growth & development Bacteria Biological and medical sciences Biology of microorganisms of confirmed or potential industrial interest Biotechnology Cloning, Molecular DNA, Bacterial - genetics Enzyme Stability Enzymology and Protein Engineering Fundamental and applied biological sciences. Psychology Genes, Bacterial Methane Methylococcus capsulatus Methylocystis Methylosinus trichosporium Microbiology Miscellaneous Mission oriented research Oxygenases - genetics Oxygenases - isolation & purification Oxygenases - metabolism Phylogeny Polymerase Chain Reaction - methods RNA, Ribosomal, 16S - genetics Sequence Analysis, DNA Solubility Substrate Specificity |
| title | Purification and characterization of the soluble methane monooxygenase of the type II methanotrophic bacterium Methylocystis sp. strain WI 14 |
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