GENE DISSECTION DEMONSTRATES THAT THE ESCHERICHIA-COLI CYSG GENE ENCODES A MULTIFUNCTIONAL PROTEIN

被引:79
作者
WARREN, MJ [1 ]
BOLT, EL [1 ]
ROESSNER, CA [1 ]
SCOTT, AI [1 ]
SPENCER, JB [1 ]
WOODCOCK, SC [1 ]
机构
[1] TEXAS A&M UNIV,CTR BIOL NMR,DEPT CHEM,COLLEGE STN,TX 77843
关键词
D O I
10.1042/bj3020837
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The C-terminus of the Escherichia coli CysG protein, consisting of amino acids 204-457, was expressed as a recombinant protein using gene dissection methodology. Analysis of the activity of this truncated protein. termed CysG(A), revealed that it was able to methylate uroporphyrinogen III in the same S-adenosyl-L-methionine (SAM)-dependent manner as the complete CysG protein. However, this truncated protein was not able to complement E. coli cysG cells, thereby suggesting that the first 201 amino acids of the CysG protein had an enzymic activity associated with the conversion of dihydrosirohydrochlorin into sirohaem. Analysis of the N-terminus of the CysG protein revealed the presence of a putative pyridine dinucleotide binding site. When the purified CysG protein was incubated with NADP(+), uroporphyrinogen III and SAM the enzyme was found to catalyse a coenzyme-mediated dehydrogenation to form sirohydrochlorin. The CysG(A) protein on the other hand showed no such coenzyme-dependent activity. Analysis of the porphyrinoid material isolated from strains harbouring plasmids containing the complete and truncated cysG genes suggested that the CysG protein was also involved in ferrochelation. The evidence presented in this paper suggests that the CysG protein is a multifunctional protein involved in SAM-dependent methylation, pyridine dinucleotide dependent dehydrogenation and ferrochelation.
引用
收藏
页码:837 / 844
页数:8
相关论文
共 25 条
[1]   PURIFICATION AND CHARACTERIZATION OF S-ADENOSYL-L-METHIONINE-UROPORPHYRINOGEN-III METHYLTRANSFERASE FROM PSEUDOMONAS-DENITRIFICANS [J].
BLANCHE, F ;
DEBUSSCHE, L ;
THIBAUT, D ;
CROUZET, J ;
CAMERON, B .
JOURNAL OF BACTERIOLOGY, 1989, 171 (08) :4222-4231
[2]   PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING OF S-ADENOSYL-L-METHIONINE - UROPORPHYRINOGEN-III METHYLTRANSFERASE FROM METHANOBACTERIUM-IVANOVII [J].
BLANCHE, F ;
ROBIN, C ;
COUDER, M ;
FAUCHER, D ;
CAUCHOIS, L ;
CAMERON, B ;
CROUZET, J .
JOURNAL OF BACTERIOLOGY, 1991, 173 (15) :4637-4645
[3]  
COLE JA, 1980, J GEN MICROBIOL, V120, P475
[4]   GENETIC AND SEQUENCE-ANALYSIS OF AN 8.7-KILOBASE PSEUDOMONAS-DENITRIFICANS FRAGMENT CARRYING 8 GENES INVOLVED IN TRANSFORMATION OF PRECORRIN-2 TO COBYRINIC ACID [J].
CROUZET, J ;
CAMERON, B ;
CAUCHOIS, L ;
RIGAULT, S ;
ROUYEZ, MC ;
BLANCHE, F ;
THIBAUT, D ;
DEBUSSCHE, L .
JOURNAL OF BACTERIOLOGY, 1990, 172 (10) :5980-5990
[5]   NUCLEOTIDE-SEQUENCE OF A PSEUDOMONAS-DENITRIFICANS 5.4-KILOBASE DNA FRAGMENT CONTAINING 5 COB GENES AND IDENTIFICATION OF STRUCTURAL GENES ENCODING S-ADENOSYL-L-METHIONINE - UROPORPHYRINOGEN-III METHYLTRANSFERASE AND COBYRINIC ACID A,C-DIAMIDE SYNTHASE [J].
CROUZET, J ;
CAUCHOIS, L ;
BLANCHE, F ;
DEBUSSCHE, L ;
THIBAUT, D ;
ROUYEZ, MC ;
RIGAULT, S ;
MAYAUX, JF ;
CAMERON, B .
JOURNAL OF BACTERIOLOGY, 1990, 172 (10) :5968-5979
[6]  
DALLEY TA, 1994, J BIOL CHEM, V269, P813
[7]   LACK OF REDOX CONTROL OF THE ANAEROBICALLY-INDUCED NIRB+ GENE OF ESCHERICHIA-COLI K-12 [J].
GRIFFITHS, L ;
COLE, JA .
ARCHIVES OF MICROBIOLOGY, 1987, 147 (04) :364-369
[8]   CLONING AND CHARACTERIZATION OF THE BACILLUS-SUBTILIS HEMEHY GENE-CLUSTER, WHICH ENCODES PROTOHEME-IX BIOSYNTHETIC-ENZYMES [J].
HANSSON, M ;
HEDERSTEDT, L .
JOURNAL OF BACTERIOLOGY, 1992, 174 (24) :8081-8093
[9]   CDNA SEQUENCE OF ADRENODOXIN REDUCTASE - IDENTIFICATION OF NADP-BINDING SITES IN OXIDOREDUCTASES [J].
HANUKOGLU, I ;
GUTFINGER, T .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1989, 180 (02) :479-484
[10]   CLONING AND SEQUENCE-ANALYSIS OF GENES INVOLVED IN ERYTHROMYCIN BIOSYNTHESIS IN SACCHAROPOLYSPORA-ERYTHRAEA - SEQUENCE SIMILARITIES BETWEEN ERYG AND A FAMILY OF S-ADENOSYLMETHIONINE-DEPENDENT METHYLTRANSFERASES [J].
HAYDOCK, SF ;
DOWSON, JA ;
DHILLON, N ;
ROBERTS, GA ;
CORTES, J ;
LEADLAY, PF .
MOLECULAR & GENERAL GENETICS, 1991, 230 (1-2) :120-128