Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (K m = 53 µM; v max = 180 nmol mg−1 min−1). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Å (R free = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D 5-symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality.

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Human GTP cyclohydrolase I: only one out of three cDNA isoforms gives rise to the active enzyme

Biochemical Journal ◽

10.1042/bj3020215 ◽

1994 ◽

Vol 302 (1) ◽

pp. 215-221 ◽

Cited By ~ 25

Author(s):

M Gütlich ◽

E Jaeger ◽

K P Rücknagel ◽

T Werner ◽

W Rödl ◽

...

Keyword(s):

Maximum Activity ◽

Open Reading Frames ◽

Intermediate Step ◽

Gtp Cyclohydrolase I ◽

Gtp Cyclohydrolase ◽

Reading Frame ◽

Cdna Sequences ◽

Rate Limiting Step ◽

Enzyme Isoforms ◽

Reading Frames

GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis. Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner. Three different cDNA forms have been reported for human GTP cyclohydrolase I [Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem. Biophys. Res. Commun. 187, 359-365]. We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al. (1992). The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli. Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate. The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction. Recombinant human GTP cyclohydrolase I showed sigmoidal substrate kinetics and maximum activity at 60 degrees C. These findings are well in line with the published properties of the enzyme isolated from rat liver. The data indicate that cytokine-mediated induction of GTP cyclohydrolase I is not due to the expression of enzyme isoforms.

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Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum

Biochemical Journal ◽

10.1042/bj3190027 ◽

1996 ◽

Vol 319 (1) ◽

pp. 27-32 ◽

Cited By ~ 9

Author(s):

Klaus WITTER ◽

Dolores J CAHILL ◽

Thomas WERNER ◽

Irmgard ZIEGLER ◽

Wolfgang RÖDL ◽

...

Keyword(s):

Dictyostelium Discoideum ◽

Single Gene ◽

Pcr Amplification ◽

Maximal Activity ◽

Chromosomal Dna ◽

Gtp Cyclohydrolase I ◽

Gtp Cyclohydrolase ◽

Reading Frame ◽

E Coli ◽

Km Value

The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 °C. The Km value for GTP is 50 µM.

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