Characterization of recombinant fructose-1,6-bisphosphate aldolase from Methylococcus capsulatus Bath
The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA...
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| Veröffentlicht in: | Biochemistry (Moscow) 2010-07, Vol.75 (7), p.892-898 |
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| creator | Rozova, O. N Khmelenina, V. N Mustakhimov, I. I Reshetnikov, A. S Trotsenko, Y. A |
| description | The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA is a hexameric enzyme (240 kDa) that is activated by Co²⁺ and inhibited by EDTA. The enzyme displays low K m to fructose-1,6-bisphosphate (FBP) and higher K m to the substrates of aldol condensation, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The FBA also catalyzes sedoheptulose-1,7-bisphosphate cleavage. The presence of Co²⁺ in the reaction mixture changes the kinetics of FBP hydrolysis and is accompanied by inhibition of the reaction by 2 mM FBP. Phylogenetically, the Mc. capsulatus enzyme belongs to the type B of class II FBAs showing high identity of translated amino acid sequence with FBAs from autotrophic bacteria. The role of the FBA in metabolism of Mc. capsulatus Bath, which realizes simultaneously three C₁ assimilating pathways (the ribulose monophosphate, the ribulose bisphosphate, and the serine cycles), is discussed. |
| doi_str_mv | 10.1134/S0006297910070114 |
| format | Article |
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N ; Khmelenina, V. N ; Mustakhimov, I. I ; Reshetnikov, A. S ; Trotsenko, Y. A</creator><creatorcontrib>Rozova, O. N ; Khmelenina, V. N ; Mustakhimov, I. I ; Reshetnikov, A. S ; Trotsenko, Y. A</creatorcontrib><description>The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA is a hexameric enzyme (240 kDa) that is activated by Co²⁺ and inhibited by EDTA. The enzyme displays low K m to fructose-1,6-bisphosphate (FBP) and higher K m to the substrates of aldol condensation, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The FBA also catalyzes sedoheptulose-1,7-bisphosphate cleavage. The presence of Co²⁺ in the reaction mixture changes the kinetics of FBP hydrolysis and is accompanied by inhibition of the reaction by 2 mM FBP. Phylogenetically, the Mc. capsulatus enzyme belongs to the type B of class II FBAs showing high identity of translated amino acid sequence with FBAs from autotrophic bacteria. The role of the FBA in metabolism of Mc. capsulatus Bath, which realizes simultaneously three C₁ assimilating pathways (the ribulose monophosphate, the ribulose bisphosphate, and the serine cycles), is discussed.</description><identifier>ISSN: 0006-2979</identifier><identifier>EISSN: 1608-3040</identifier><identifier>DOI: 10.1134/S0006297910070114</identifier><identifier>PMID: 20673213</identifier><language>eng</language><publisher>Dordrecht: Dordrecht : SP MAIK Nauka/Interperiodica</publisher><subject>Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - isolation & purification ; Bacterial Proteins - metabolism ; Biochemistry ; Biomedical and Life Sciences ; Biomedicine ; Bioorganic Chemistry ; Enzyme Stability ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Fructose-Bisphosphate Aldolase - chemistry ; Fructose-Bisphosphate Aldolase - genetics ; Fructose-Bisphosphate Aldolase - isolation & purification ; Fructose-Bisphosphate Aldolase - metabolism ; Gene Expression ; Kinetics ; Life Sciences ; Methylococcus capsulatus - chemistry ; Methylococcus capsulatus - classification ; Methylococcus capsulatus - enzymology ; Microbiology ; Molecular Sequence Data ; Molecular Weight ; Phylogeny ; Protein Multimerization ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Substrate Specificity</subject><ispartof>Biochemistry (Moscow), 2010-07, Vol.75 (7), p.892-898</ispartof><rights>Pleiades Publishing, Ltd. 2010</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c367t-b199e160a822f0abf35ba8eacb6c5202381acc4c7453a94421b1208d3cb8f2b63</citedby><cites>FETCH-LOGICAL-c367t-b199e160a822f0abf35ba8eacb6c5202381acc4c7453a94421b1208d3cb8f2b63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1134/S0006297910070114$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1134/S0006297910070114$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27903,27904,41467,42536,51298</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20673213$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Rozova, O. N</creatorcontrib><creatorcontrib>Khmelenina, V. N</creatorcontrib><creatorcontrib>Mustakhimov, I. I</creatorcontrib><creatorcontrib>Reshetnikov, A. S</creatorcontrib><creatorcontrib>Trotsenko, Y. A</creatorcontrib><title>Characterization of recombinant fructose-1,6-bisphosphate aldolase from Methylococcus capsulatus Bath</title><title>Biochemistry (Moscow)</title><addtitle>Biochemistry Moscow</addtitle><addtitle>Biochemistry (Mosc)</addtitle><description>The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA is a hexameric enzyme (240 kDa) that is activated by Co²⁺ and inhibited by EDTA. The enzyme displays low K m to fructose-1,6-bisphosphate (FBP) and higher K m to the substrates of aldol condensation, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The FBA also catalyzes sedoheptulose-1,7-bisphosphate cleavage. The presence of Co²⁺ in the reaction mixture changes the kinetics of FBP hydrolysis and is accompanied by inhibition of the reaction by 2 mM FBP. Phylogenetically, the Mc. capsulatus enzyme belongs to the type B of class II FBAs showing high identity of translated amino acid sequence with FBAs from autotrophic bacteria. The role of the FBA in metabolism of Mc. capsulatus Bath, which realizes simultaneously three C₁ assimilating pathways (the ribulose monophosphate, the ribulose bisphosphate, and the serine cycles), is discussed.</description><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - isolation & purification</subject><subject>Bacterial Proteins - metabolism</subject><subject>Biochemistry</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>Bioorganic Chemistry</subject><subject>Enzyme Stability</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Fructose-Bisphosphate Aldolase - chemistry</subject><subject>Fructose-Bisphosphate Aldolase - genetics</subject><subject>Fructose-Bisphosphate Aldolase - isolation & purification</subject><subject>Fructose-Bisphosphate Aldolase - metabolism</subject><subject>Gene Expression</subject><subject>Kinetics</subject><subject>Life Sciences</subject><subject>Methylococcus capsulatus - chemistry</subject><subject>Methylococcus capsulatus - classification</subject><subject>Methylococcus capsulatus - enzymology</subject><subject>Microbiology</subject><subject>Molecular Sequence Data</subject><subject>Molecular Weight</subject><subject>Phylogeny</subject><subject>Protein Multimerization</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Substrate Specificity</subject><issn>0006-2979</issn><issn>1608-3040</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9ULtOKzEQtRAIcgMfQAPb0dyFGdvZRwkRcJGCKIDaGjtesmh3HWxvAV-PowAN0i1GM6PzkM5h7BjhHFHIi0cAKHhd1ghQAqLcYRMsoMoFSNhlkw2cb_AD9ieE1_RyqMU-O-BQlIKjmDA7X5EnE61vPyi2bshck3lrXK_bgYaYNX400QWb498i121Yr1waijajbuk6CjZRXJ_d27h675xxxowhM7QOY0cxnVcUV4dsr6Eu2KOvPWXPN9dP83_54uH2bn65yI0oyphrrGubAlDFeQOkGzHTVFkyujAzDlxUSMZIU8qZoFpKjho5VEthdNVwXYgpO9v6rr17G22Iqm-DsV1Hg3VjUKWsai7rVMKU4ZZpvAvB20atfduTf1cIalOu-lVu0px8uY-6t8sfxXebicC3hJCg4cV69epGP6TE_3U93YoacopefBvU8yMHFIBVCXJWik-xgo3e</recordid><startdate>20100701</startdate><enddate>20100701</enddate><creator>Rozova, O. 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A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c367t-b199e160a822f0abf35ba8eacb6c5202381acc4c7453a94421b1208d3cb8f2b63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - isolation & purification</topic><topic>Bacterial Proteins - metabolism</topic><topic>Biochemistry</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedicine</topic><topic>Bioorganic Chemistry</topic><topic>Enzyme Stability</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Fructose-Bisphosphate Aldolase - chemistry</topic><topic>Fructose-Bisphosphate Aldolase - genetics</topic><topic>Fructose-Bisphosphate Aldolase - isolation & purification</topic><topic>Fructose-Bisphosphate Aldolase - metabolism</topic><topic>Gene Expression</topic><topic>Kinetics</topic><topic>Life Sciences</topic><topic>Methylococcus capsulatus - chemistry</topic><topic>Methylococcus capsulatus - classification</topic><topic>Methylococcus capsulatus - enzymology</topic><topic>Microbiology</topic><topic>Molecular Sequence Data</topic><topic>Molecular Weight</topic><topic>Phylogeny</topic><topic>Protein Multimerization</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Rozova, O. N</creatorcontrib><creatorcontrib>Khmelenina, V. N</creatorcontrib><creatorcontrib>Mustakhimov, I. I</creatorcontrib><creatorcontrib>Reshetnikov, A. S</creatorcontrib><creatorcontrib>Trotsenko, Y. A</creatorcontrib><collection>AGRIS</collection><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><jtitle>Biochemistry (Moscow)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Rozova, O. N</au><au>Khmelenina, V. N</au><au>Mustakhimov, I. I</au><au>Reshetnikov, A. S</au><au>Trotsenko, Y. A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of recombinant fructose-1,6-bisphosphate aldolase from Methylococcus capsulatus Bath</atitle><jtitle>Biochemistry (Moscow)</jtitle><stitle>Biochemistry Moscow</stitle><addtitle>Biochemistry (Mosc)</addtitle><date>2010-07-01</date><risdate>2010</risdate><volume>75</volume><issue>7</issue><spage>892</spage><epage>898</epage><pages>892-898</pages><issn>0006-2979</issn><eissn>1608-3040</eissn><abstract>The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA is a hexameric enzyme (240 kDa) that is activated by Co²⁺ and inhibited by EDTA. The enzyme displays low K m to fructose-1,6-bisphosphate (FBP) and higher K m to the substrates of aldol condensation, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The FBA also catalyzes sedoheptulose-1,7-bisphosphate cleavage. The presence of Co²⁺ in the reaction mixture changes the kinetics of FBP hydrolysis and is accompanied by inhibition of the reaction by 2 mM FBP. Phylogenetically, the Mc. capsulatus enzyme belongs to the type B of class II FBAs showing high identity of translated amino acid sequence with FBAs from autotrophic bacteria. The role of the FBA in metabolism of Mc. capsulatus Bath, which realizes simultaneously three C₁ assimilating pathways (the ribulose monophosphate, the ribulose bisphosphate, and the serine cycles), is discussed.</abstract><cop>Dordrecht</cop><pub>Dordrecht : SP MAIK Nauka/Interperiodica</pub><pmid>20673213</pmid><doi>10.1134/S0006297910070114</doi><tpages>7</tpages></addata></record> |
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| subjects | Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - isolation & purification Bacterial Proteins - metabolism Biochemistry Biomedical and Life Sciences Biomedicine Bioorganic Chemistry Enzyme Stability Escherichia coli - genetics Escherichia coli - metabolism Fructose-Bisphosphate Aldolase - chemistry Fructose-Bisphosphate Aldolase - genetics Fructose-Bisphosphate Aldolase - isolation & purification Fructose-Bisphosphate Aldolase - metabolism Gene Expression Kinetics Life Sciences Methylococcus capsulatus - chemistry Methylococcus capsulatus - classification Methylococcus capsulatus - enzymology Microbiology Molecular Sequence Data Molecular Weight Phylogeny Protein Multimerization Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Substrate Specificity |
| title | Characterization of recombinant fructose-1,6-bisphosphate aldolase from Methylococcus capsulatus Bath |
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