An O-Acetylserine (thiol) Lyase from Leucaena leucocephala Is a Cysteine Synthase But Not a Mimosine Synthase
In plants, the final step of cysteine formation is catalyzed by O -acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also...
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| Veröffentlicht in: | Applied biochemistry and biotechnology 2014-07, Vol.173 (5), p.1157-1168 |
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| creator | Yafuso, Jannai T. Negi, Vishal Singh Bingham, Jon-Paul Borthakur, Dulal |
| description | In plants, the final step of cysteine formation is catalyzed by
O
-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume
Leucaena leucocephala
(leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from
Escherichia coli
and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the
K
m
was calculated to be 1,850 ± 414 μM sulfide and the
V
max
was 200.6 ± 19.92 μM cysteine min
−1
. The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as β-substituted alanine synthases. |
| doi_str_mv | 10.1007/s12010-014-0917-z |
| format | Article |
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O
-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume
Leucaena leucocephala
(leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from
Escherichia coli
and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the
K
m
was calculated to be 1,850 ± 414 μM sulfide and the
V
max
was 200.6 ± 19.92 μM cysteine min
−1
. The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as β-substituted alanine synthases.</description><identifier>ISSN: 0273-2289</identifier><identifier>EISSN: 1559-0291</identifier><identifier>DOI: 10.1007/s12010-014-0917-z</identifier><identifier>PMID: 24777760</identifier><identifier>CODEN: ABIBDL</identifier><language>eng</language><publisher>Boston: Springer US</publisher><subject>Amino acids ; Binding sites ; Biochemistry ; Biological and medical sciences ; Biotechnology ; Chemistry ; Chemistry and Materials Science ; Cysteine - biosynthesis ; Cysteine Synthase - genetics ; Cysteine Synthase - isolation & purification ; Cysteine Synthase - metabolism ; E coli ; Enzymatic activity ; Enzyme kinetics ; Enzymes ; Escherichia coli ; Escherichia coli - genetics ; Fabaceae - enzymology ; Fodder ; Fundamental and applied biological sciences. Psychology ; Legumes ; Leucaena ; Leucaena leucocephala ; Mimosine - metabolism ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Substrates</subject><ispartof>Applied biochemistry and biotechnology, 2014-07, Vol.173 (5), p.1157-1168</ispartof><rights>Springer Science+Business Media New York 2014</rights><rights>2015 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c468t-b32912c480b0b0b33c6e33ec3353fc82c3b00107ac95017b50bc8b3a18c088d63</citedby><cites>FETCH-LOGICAL-c468t-b32912c480b0b0b33c6e33ec3353fc82c3b00107ac95017b50bc8b3a18c088d63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s12010-014-0917-z$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s12010-014-0917-z$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=28677168$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24777760$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Yafuso, Jannai T.</creatorcontrib><creatorcontrib>Negi, Vishal Singh</creatorcontrib><creatorcontrib>Bingham, Jon-Paul</creatorcontrib><creatorcontrib>Borthakur, Dulal</creatorcontrib><title>An O-Acetylserine (thiol) Lyase from Leucaena leucocephala Is a Cysteine Synthase But Not a Mimosine Synthase</title><title>Applied biochemistry and biotechnology</title><addtitle>Appl Biochem Biotechnol</addtitle><addtitle>Appl Biochem Biotechnol</addtitle><description>In plants, the final step of cysteine formation is catalyzed by
O
-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume
Leucaena leucocephala
(leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from
Escherichia coli
and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the
K
m
was calculated to be 1,850 ± 414 μM sulfide and the
V
max
was 200.6 ± 19.92 μM cysteine min
−1
. The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as β-substituted alanine synthases.</description><subject>Amino acids</subject><subject>Binding sites</subject><subject>Biochemistry</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Cysteine - biosynthesis</subject><subject>Cysteine Synthase - genetics</subject><subject>Cysteine Synthase - isolation & purification</subject><subject>Cysteine Synthase - metabolism</subject><subject>E coli</subject><subject>Enzymatic activity</subject><subject>Enzyme kinetics</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Fabaceae - enzymology</subject><subject>Fodder</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Legumes</subject><subject>Leucaena</subject><subject>Leucaena leucocephala</subject><subject>Mimosine - metabolism</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Substrates</subject><issn>0273-2289</issn><issn>1559-0291</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp1kV9P5CAUxclGs466H2BfDInZRB9YL9AW-jg78V8yrg_qM6FIdzppywjtQ_300nRcJybCAyT3d7iXcxD6SeE3BRAXgTKgQIAmBHIqyOs3NKNpmhNgOd1DM2CCE8ZkfoAOQ1gDUCZT8R0dsETElcEMNfMW35O5sd1QB-ur1uKzblW5-hwvBx0sLr1r8NL2RttW4zpenLGbla41vg1Y48UQOjvKHoa2W42KP32H_7ou1u6qxoXd2jHaL3Xs82N7HqGnq8vHxQ1Z3l_fLuZLYpJMdqTgcX5mEgnFuDk3meXcGs5TXhrJDC_iX0Bok6dARZFCYWTBNZUGpHzO-BE6m97dePfS29CppgrG1rVureuDiialoxmCRvT0E7p2vW_jdJFK2GhTLiNFJ8p4F4K3pdr4qtF-UBTUmIWaslAxCzVmoV6j5mT7cl809vm_4t38CPzaAjoYXZdet6YKH5zMhKDZ2JxNXIil9p_1OyN-2f0NkiGfxw</recordid><startdate>20140701</startdate><enddate>20140701</enddate><creator>Yafuso, Jannai T.</creator><creator>Negi, Vishal Singh</creator><creator>Bingham, Jon-Paul</creator><creator>Borthakur, Dulal</creator><general>Springer US</general><general>Springer</general><general>Springer Nature B.V</general><scope>IQODW</scope><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>3V.</scope><scope>7ST</scope><scope>7T7</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>RC3</scope><scope>SOI</scope><scope>7QO</scope></search><sort><creationdate>20140701</creationdate><title>An O-Acetylserine (thiol) Lyase from Leucaena leucocephala Is a Cysteine Synthase But Not a Mimosine Synthase</title><author>Yafuso, Jannai T. ; Negi, Vishal Singh ; Bingham, Jon-Paul ; Borthakur, Dulal</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c468t-b32912c480b0b0b33c6e33ec3353fc82c3b00107ac95017b50bc8b3a18c088d63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Amino acids</topic><topic>Binding sites</topic><topic>Biochemistry</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Cysteine - biosynthesis</topic><topic>Cysteine Synthase - genetics</topic><topic>Cysteine Synthase - isolation & purification</topic><topic>Cysteine Synthase - metabolism</topic><topic>E coli</topic><topic>Enzymatic activity</topic><topic>Enzyme kinetics</topic><topic>Enzymes</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Fabaceae - enzymology</topic><topic>Fodder</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Legumes</topic><topic>Leucaena</topic><topic>Leucaena leucocephala</topic><topic>Mimosine - metabolism</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Substrates</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Yafuso, Jannai T.</creatorcontrib><creatorcontrib>Negi, Vishal Singh</creatorcontrib><creatorcontrib>Bingham, Jon-Paul</creatorcontrib><creatorcontrib>Borthakur, Dulal</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>ProQuest Central (Corporate)</collection><collection>Environment Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>Environment Abstracts</collection><collection>Biotechnology Research Abstracts</collection><jtitle>Applied biochemistry and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Yafuso, Jannai T.</au><au>Negi, Vishal Singh</au><au>Bingham, Jon-Paul</au><au>Borthakur, Dulal</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An O-Acetylserine (thiol) Lyase from Leucaena leucocephala Is a Cysteine Synthase But Not a Mimosine Synthase</atitle><jtitle>Applied biochemistry and biotechnology</jtitle><stitle>Appl Biochem Biotechnol</stitle><addtitle>Appl Biochem Biotechnol</addtitle><date>2014-07-01</date><risdate>2014</risdate><volume>173</volume><issue>5</issue><spage>1157</spage><epage>1168</epage><pages>1157-1168</pages><issn>0273-2289</issn><eissn>1559-0291</eissn><coden>ABIBDL</coden><abstract>In plants, the final step of cysteine formation is catalyzed by
O
-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume
Leucaena leucocephala
(leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from
Escherichia coli
and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the
K
m
was calculated to be 1,850 ± 414 μM sulfide and the
V
max
was 200.6 ± 19.92 μM cysteine min
−1
. The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as β-substituted alanine synthases.</abstract><cop>Boston</cop><pub>Springer US</pub><pmid>24777760</pmid><doi>10.1007/s12010-014-0917-z</doi><tpages>12</tpages></addata></record> |
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| ispartof | Applied biochemistry and biotechnology, 2014-07, Vol.173 (5), p.1157-1168 |
| issn | 0273-2289 1559-0291 |
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| source | MEDLINE; SpringerLink Journals - AutoHoldings |
| subjects | Amino acids Binding sites Biochemistry Biological and medical sciences Biotechnology Chemistry Chemistry and Materials Science Cysteine - biosynthesis Cysteine Synthase - genetics Cysteine Synthase - isolation & purification Cysteine Synthase - metabolism E coli Enzymatic activity Enzyme kinetics Enzymes Escherichia coli Escherichia coli - genetics Fabaceae - enzymology Fodder Fundamental and applied biological sciences. Psychology Legumes Leucaena Leucaena leucocephala Mimosine - metabolism Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Substrates |
| title | An O-Acetylserine (thiol) Lyase from Leucaena leucocephala Is a Cysteine Synthase But Not a Mimosine Synthase |
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