BIOSYNTHESIS OF CAPSULAR POLYSACCHARIDES OF PATHOGENIC BACTERIA
BIOSYNTHESIS OF CAPSULAR POLYSACCHARIDES OF PATHOGENIC BACTERIA
批准号:
3792306
负责人:
W F VANN
金额:
$0.0万
依托单位:
--
依托单位国家:
美国
项目类别:
财政年份:
--
资助国家:
美国
项目状态:
未结题
起止时间:
至
关键词:
Escherichia coli N acetylneuraminate bacterial antigens bacterial capsules bacterial polysaccharides bacterial proteins carbohydrate biosynthesis chemical kinetics cysteine cytidine enzyme activity enzyme mechanism enzyme structure glycosyltransferase maleimides microorganism metabolism nucleotidyltransferase protein purification protein sequence site directed mutagenesis
中文摘要
大肠杆菌CMP-NeuAc合成酶催化胞苷5'的形成
英文摘要
Escherichia coli CMP-NeuAc synthetase catalyses formation of cytidine 5'
monophospho-N-acetylneuraminic acid (CMP-NeuAc) from N-acetylneuraminic
acid (NeuAc) and cytidine triphosphate (CTP). Availability and specificity
of purified sugar activating enzymes and glycosyltransferases make these
enzymes convenient tools for synthesis of biologically relevant
oligosaccharides. The role of the cysteines in the structural and
catalytic properties of the enzyme has been examined by site directed
mutagenesis and chemical modification. Chemical modification with the
sulfhydro specific reagent DTDP suggests that one cysteine residue is
involved in catalysis, since that enzyme can be completely inactivated by
titration of one cysteine residue with DTDP. The enzyme can be protected
from inactivation in the presence of the substrate CTP. Site directed
mutagenesis demonstrates that cysteine 129 and cysteine 329 are not
essential for catalysis, since both can be substituted by selected amino
acids without complete loss of activity. Chemical inactivation with N-
ethylmaleimide (NEM) demonstrates that cysteine 329 and cysteine 129 are
inaccessible to NEM at room temperature. Cysteine 329 can be thermally
exposed to chemical modification with NEM at 42 degree C but cysteine 129
is inaccessible to chemical modification with NEM. We have sequenced the
amino terminus of the CMP NeuAc synthetase from meningococcus group B which
synthesizes the same a 2-8 NeuAc capsule of E. coli K 1. The N terminal
sequences were compared and a concensus obtained. Arginine and lysine
residues were identified as common amino acids in the compared sequences.
We have mutated 14 arginines in E. coli CMP NeuAc synthetase to Glycine and
mutation of the common arginine completely inactivates CMP NeuAc
synthetase. Changing arginine to alanine results in an inactivated enzyme,
but a change to lysine restores the specific activity to wild type values.
Km determinations are being done at different pHs to determine involvement
of arginine in catalysis.
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STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN THE METABOLISM OF POLYSIALIC ACID
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批准号:2336399
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负责人:W F VANN
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CHEMICAL SYNTHESIS AND STRUCTURE OF BACTERIAL POLYSACCHARIDES AND CONJUGATES
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批准号:2568879
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负责人:W F VANN
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STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN THE METABOLISM OF POLYSIALIC ACID
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批准号:3748111
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负责人:W F VANN
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CHEMICAL SYNTHESIS AND STRUCTURE OF BACTERIAL POLYSACCHARIDES
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批准号:3770282
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负责人:W F VANN
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STRUCTURE AND IMMUNOCHEMISTRY OF CAPSULAR POLYSACCHARIDES
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批准号:3804609
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资助金额:$0.0万
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负责人:W F VANN
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STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN THE METABOLISM OF POLYSIALIC ACID
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批准号:6161192
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负责人:W F VANN
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BIOSYNTHESIS OF CAPSULAR POLYSACCHARIDES OF PATHOGENIC BACTERIA
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批准号:3804594
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负责人:W F VANN
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STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN THE METABO
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批准号:6546984
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资助金额:$0.0万
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负责人:W F VANN
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STRUCTURE/FUNCTION RELATIONSHIPS OF DIPHTHERIA TOXIN A CHAIN
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批准号:6161210
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资助金额:$0.0万
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负责人:W F VANN
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CHEMICAL SYNTHESIS AND STRUCTURE OF BACTERIAL POLYSACCHARIDES
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批准号:3748110
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资助金额:$0.0万
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负责人:W F VANN
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CHEMICAL SYNTHESIS OF MENINGOCOCCAL GROUP A OLIGOSACCHARIDES
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批准号:3792317
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负责人:W F VANN
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BIOSYNTHESIS OF CAPSULAR POLYSACCHARIDES OF PATHOGENIC BACTERIA
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批准号:3810962
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负责人:W F VANN
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CHEMICAL SYNTHESIS AND STRUCTURE OF BACTERIAL POLYSACCHARIDES AND CONJUGATES
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批准号:6161191
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负责人:W F VANN
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CHEMICAL SYNTHESIS AND STRUCTURE OF BACTERIAL POLYSACCHA
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批准号:6546981
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资助金额:$0.0万
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负责人:W F VANN
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STRUCTURE/FUNCTION RELATIONSHIPS OF DIPHTHERIA TOXIN A C
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批准号:6547054
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资助金额:$0.0万
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负责人:W F VANN
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依托单位:--
CHEMICAL SYNTHESIS AND STRUCTURE OF BACTERIAL POLYSACCHARIDES AND CONJUGATES
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批准号:5200677
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资助金额:$0.0万
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负责人:W F VANN
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STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN THE METABOLISM OF POLYSIALIC ACID
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批准号:3770283
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项目类别:
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资助金额:$0.0万
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负责人:W F VANN
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