CDP-6-DEOXY-DELTA(3)(4)-GLUCOSEEN REDUCTASE FROM YERSINIA-PSEUDOTUBERCULOSIS - ENZYME-PURIFICATION AND CHARACTERIZATION OF THE CLONED GENE

被引:37
作者
LO, SF
MILLER, VP
LEI, YY
THORSON, JS
LIU, HW
SCHOTTEL, JL
机构
[1] UNIV MINNESOTA,DEPT CHEM,KOLTHOFF & SMITH HALLS,207 PLEASANT ST SE,MINNEAPOLIS,MN 55455
[2] UNIV MINNESOTA,DEPT BIOCHEM,ST PAUL,MN 55108
关键词
D O I
10.1128/JB.176.2.460-468.1994
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The 3,6-dideoxyhexoses, usually confined to the cell wall lipopolysaccharide of gram-negative bacteria, are essential to serological specificity and are formed via a complex biosynthetic pathway beginning with CDP-D-hexoses. In particular, the biosynthesis of CDP-ascarylose, one of the naturally occurring 3,6-dideoxyhexoses, consists of five enzymatic steps, with CDP-6-deoxy-DELTA3,4-glucoseen reductase (E3) participating as the key enzyme in this catalysis. This enzyme has been previously purified from Yersinia pseudotuberculosis by an unusual procedure (protocol 1) including a trypsin digestion step (O. Han, V. P. Miller, and H.-W. Liu, J. Biol. Chem. 265:8033-8041, 1990). However, the cloned gene showed disparity with the expected gene characteristics, and upon expression, the resulting gene product exhibited no E3 activity. These findings strongly suggested that the protein isolated by protocol I may have been misidentified as E3. A reinvestigation of the purification protocol produced a new and improved procedure (protocol 11) consisting of DEAE-Sephacel, phenyl-Sepharose, Cibacron blue A, and Sephadex G-100 chromatography, which efficiently yielded a new homogeneous enzyme composed of a single polypeptide with a molecular weight of 39,000. This highly purified protein had a specific activity nearly 8,000-fold higher than that of cell lysates, and more importantly, the corresponding gene (ascD) was found to be part of the ascarylose biosynthetic cluster. Presented are the identification and confirmation of the E3 gene through cloning and overexpression and the culminating purification and unambiguous assignment of homogeneous E3. The nucleotide and translated amino acid sequences of the genuine E3 are also presented.
引用
收藏
页码:460 / 468
页数:9
相关论文
共 49 条
[1]   ESTIMATION OF MOLECULAR WEIGHTS OF PROTEINS BY SEPHADEX GEL-FILTRATION [J].
ANDREWS, P .
BIOCHEMICAL JOURNAL, 1964, 91 (02) :222-+
[2]  
ASHWELL G, 1971, MICROBIAL TOXINS, V4, P235
[3]  
AUSUBEL FM, 1989, CURRENT PROTOCOLS MO
[4]  
BIRNBOIM HC, 1979, NUCLEIC ACIDS RES, V7, P1513
[5]  
BISHOP CT, 1982, POLYSACCHARIDES, V1, P291
[6]  
BRUTLAG DL, 1990, COMPUT APPL BIOSCI, V6, P237
[7]  
Butterworth R. F., 1971, ADV CARBOHYD CHEM BI, V26, P279
[8]   ESTIMATION OF 3-DEOXY SUGARS BY MEANS OF THE MALONALDEHYDE-THIOBARBITURIC ACID REACTION [J].
CYNKIN, MA ;
ASHWELL, G .
NATURE, 1960, 186 (4719) :155-156
[9]  
DAWSON RMC, 1986, DATA BIOCH RES, P355
[10]  
GLASER L, 1971, ENZYMES, V5, P465