IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis.
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 dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein 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 microM, 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 the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.
Gräwert
Tobias
T
Contribution from the Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Kaiser
Johannes
J
Zepeck
Ferdinand
F
Laupitz
Ralf
R
Hecht
Stefan
S
Amslinger
Sabine
S
Schramek
Nicholas
N
Schleicher
Erik
E
Weber
Stefan
S
Haslbeck
Martin
M
Buchner
Johannes
J
Rieder
Christoph
C
Arigoni
Duilio
D
Bacher
Adelbert
A
Eisenreich
Wolfgang
W
Rohdich
Felix
F
D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C109866:ispH protein, E coli