Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase
The Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in Escherichia coli. The preparation yields 470 UL −1 of enzyme at a specific activity of 9.4 U mg −1. During retroaldol cleavage of KDPG, the enzyme shows a k cat that decreases with decreasing tempe...
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| Veröffentlicht in: | Bioorganic & medicinal chemistry 2002-03, Vol.10 (3), p.545-550 |
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| container_title | Bioorganic & medicinal chemistry |
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| creator | Griffiths, Jennifer S Wymer, Nathan J Njolito, Eugenia Niranjanakumari, S Fierke, Carol A Toone, Eric J |
| description | The
Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in
Escherichia coli. The preparation yields 470
UL
−1 of enzyme at a specific activity of 9.4
U
mg
−1. During retroaldol cleavage of KDPG, the enzyme shows a
k
cat that decreases with decreasing temperature. A more than offsetting decrease in
K
m yields an enzyme that is more efficient at 40
°C than at 70
°C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.
Graphic |
| doi_str_mv | 10.1016/S0968-0896(01)00307-8 |
| format | Article |
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Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in
Escherichia coli. The preparation yields 470
UL
−1 of enzyme at a specific activity of 9.4
U
mg
−1. During retroaldol cleavage of KDPG, the enzyme shows a
k
cat that decreases with decreasing temperature. A more than offsetting decrease in
K
m yields an enzyme that is more efficient at 40
°C than at 70
°C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.
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Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in
Escherichia coli. The preparation yields 470
UL
−1 of enzyme at a specific activity of 9.4
U
mg
−1. During retroaldol cleavage of KDPG, the enzyme shows a
k
cat that decreases with decreasing temperature. A more than offsetting decrease in
K
m yields an enzyme that is more efficient at 40
°C than at 70
°C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.
Graphic</description><subject>Aldehyde-Lyases - genetics</subject><subject>Aldehyde-Lyases - isolation & purification</subject><subject>Aldehyde-Lyases - metabolism</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Cloning, Molecular</subject><subject>Enzyme engineering</subject><subject>Escherichia coli - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gluconates - metabolism</subject><subject>Kinetics</subject><subject>Methods. Procedures. Technologies</subject><subject>Miscellaneous</subject><subject>Substrate Specificity</subject><subject>Temperature</subject><subject>Thermotoga maritima - enzymology</subject><issn>0968-0896</issn><issn>1464-3391</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkE1P3DAQhi1UVBboT2iVSyuQSBknTmKfKrR8qiuxEsvZcuwx6yqJwc4iwa_HsCv22NNIo-edefUQ8p3Cbwq0Pr0DUfMcuKiPgB4DlNDkfIdMKKtZXpaCfiGTT2SP7Mf4DwAKJuhXskcpp4wzmJD5tPODGx5OMhd9p0bnh0wNJtNLFZQeMbjX9dLbbFxitlhi6P3oH1TWq-BG16vs7_n8KlOdSfmIh2TXqi7it808IPeXF4vpdT67vbqZns1ynbqNuYFWNbSyAFVbNKxhFGreahCisIIrwS0KYw3WrS6qQrdlyxBAcKxsZdKF8oD8Wt99DP5phXGUvYsau04N6FdRNpTRInlIYLUGdfAxBrTyMaTW4UVSkO8q5YdK-e5JApUfKiVPuR-bB6u2R7NNbdwl4OcGUFGrzgY1aBe3XMmKpimaxP1Zc5h0PDsMMmqHg0bjAupRGu_-U-UN-6WP6w</recordid><startdate>20020301</startdate><enddate>20020301</enddate><creator>Griffiths, Jennifer S</creator><creator>Wymer, Nathan J</creator><creator>Njolito, Eugenia</creator><creator>Niranjanakumari, S</creator><creator>Fierke, Carol A</creator><creator>Toone, Eric J</creator><general>Elsevier Ltd</general><general>Elsevier Science</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>7X8</scope></search><sort><creationdate>20020301</creationdate><title>Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase</title><author>Griffiths, Jennifer S ; Wymer, Nathan J ; Njolito, Eugenia ; Niranjanakumari, S ; Fierke, Carol A ; Toone, Eric J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c391t-d0ba715f005b274741068bc0992f98a98fe9dfde6bc252cb3b4e0098e5f5d3913</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Aldehyde-Lyases - genetics</topic><topic>Aldehyde-Lyases - isolation & purification</topic><topic>Aldehyde-Lyases - metabolism</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Cloning, Molecular</topic><topic>Enzyme engineering</topic><topic>Escherichia coli - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gluconates - metabolism</topic><topic>Kinetics</topic><topic>Methods. Procedures. Technologies</topic><topic>Miscellaneous</topic><topic>Substrate Specificity</topic><topic>Temperature</topic><topic>Thermotoga maritima - enzymology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Griffiths, Jennifer S</creatorcontrib><creatorcontrib>Wymer, Nathan J</creatorcontrib><creatorcontrib>Njolito, Eugenia</creatorcontrib><creatorcontrib>Niranjanakumari, S</creatorcontrib><creatorcontrib>Fierke, Carol A</creatorcontrib><creatorcontrib>Toone, Eric J</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>MEDLINE - Academic</collection><jtitle>Bioorganic & medicinal chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Griffiths, Jennifer S</au><au>Wymer, Nathan J</au><au>Njolito, Eugenia</au><au>Niranjanakumari, S</au><au>Fierke, Carol A</au><au>Toone, Eric J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase</atitle><jtitle>Bioorganic & medicinal chemistry</jtitle><addtitle>Bioorg Med Chem</addtitle><date>2002-03-01</date><risdate>2002</risdate><volume>10</volume><issue>3</issue><spage>545</spage><epage>550</epage><pages>545-550</pages><issn>0968-0896</issn><eissn>1464-3391</eissn><abstract>The
Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in
Escherichia coli. The preparation yields 470
UL
−1 of enzyme at a specific activity of 9.4
U
mg
−1. During retroaldol cleavage of KDPG, the enzyme shows a
k
cat that decreases with decreasing temperature. A more than offsetting decrease in
K
m yields an enzyme that is more efficient at 40
°C than at 70
°C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.
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| fulltext | fulltext |
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| ispartof | Bioorganic & medicinal chemistry, 2002-03, Vol.10 (3), p.545-550 |
| issn | 0968-0896 1464-3391 |
| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Aldehyde-Lyases - genetics Aldehyde-Lyases - isolation & purification Aldehyde-Lyases - metabolism Biological and medical sciences Biotechnology Cloning, Molecular Enzyme engineering Escherichia coli - genetics Fundamental and applied biological sciences. Psychology Gluconates - metabolism Kinetics Methods. Procedures. Technologies Miscellaneous Substrate Specificity Temperature Thermotoga maritima - enzymology |
| title | Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase |
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