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
N-乙酰神经氨酸的单磷-N-乙酰神经氨酸(CMP-NeuAc)
酸(NeuAc)和胞苷三磷酸(CTP)。可用性和专用性
纯化的糖激活酶和糖基转移酶使这些
酶是合成生物相关物质的便捷工具
低聚糖。半胱氨酸在结构和代谢中的作用
酶的催化性质用定点方法进行了考察
诱变和化学修饰。用化学方法进行修饰
磺基特异性试剂DTDP表明,一个半胱氨酸残基是
参与催化,因为这种酶可以完全失活
用DTDP滴定一种半胱氨酸残留量。这种酶可以被保护起来
在底物CTP存在的情况下使其失活。站点定向
诱变表明半胱氨酸129和半胱氨酸329不是
对催化是必不可少的,因为两者都可以被选定的氨基取代
酸而不会完全失去活性。N-的化学灭活
乙基马来酰亚胺(NEM)证明半胱氨酸329和半胱氨酸129是
NEM在室温下无法接触到。半胱氨酸329可以在热
在42摄氏度下暴露于NEM的化学修饰中,但半胱氨酸129
不能用NEM进行化学修饰。我们已经测序了
B组脑膜炎双球菌CMPNeuAc合成酶的氨基端
合成了同样的大肠杆菌K1的2-8NeuAc胶囊。N端
对序列进行了比较,得到了一致的结果。精氨酸和赖氨酸
在比较的序列中,残基被鉴定为常见的氨基酸。
我们已经将E.ColiCMPNeuAc合成酶中的14种精氨酸突变为甘氨酸和
普通精氨酸的突变完全失活cMP NeuAc
合成酶。把精氨酸变成丙氨酸会导致失活的酶,
但改变为赖氨酸可以将比活性恢复到野生型的值。
KM测定正在不同的PHS进行,以确定是否参与其中
精氨酸在催化中的作用。
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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资助金额:$0.0万
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资助金额:$0.0万
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