IspH Protein of Escherichia coli: Studies on Iron-Sulfur Cluster Implementation and Catalysis
详细信息 查看全文
- 作者：Tobias Grä ; wert ; Johannes Kaiser ; Ferdinand Zepeck ; Ralf Laupitz ; Stefan Hecht ; Sabine Amslinger ; Nicholas Schramek ; Erik Schleicher ; Stefan Weber ; Martin Haslbeck ; Johannes Buchner ; Christoph Rie
- 刊名：Journal of the American Chemical Society
- 出版年：2004
- 出版时间：October 13, 2004
- 年：2004
- 卷：126
- 期：40
- 页码：12847 - 12855
- 全文大小：147K
- 年卷期：v.126,no.40(October 13, 2004)
- ISSN：1520-5126
文摘
The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyldiphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of agene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clustersinto an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspHprotein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity,flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 
M, respectively.EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S]+ cluster in IspH protein.Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at theamino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced thecatalytic activity by a factor of more than 70 000.
M, respectively.EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S]+ cluster in IspH protein.Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at theamino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced thecatalytic activity by a factor of more than 70 000.