Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I.
The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass of 24,563Da. The uncommon start codon TTG was identified by matching the N-terminal amino acid sequence of purified Nocardia GCH with the deduced amino acid sequence. A likely ribosomal binding site was identified 9bp upstream of the translational start site. The 3' end flank region encodes a peptide that shares high homology with dihydropteroate synthases. Nocardia GCH has 73 and 60% identity to the proteins encoded by the putative gch of Mycobacterium tuberculosis and Streptomyces coelicolor, respectively. Nocardia GCH was highly expressed in Escherichia coli cells carrying a pHAT10 based expression vector, and moderately expressed in Mycobacterium smegmatis cells carrying a pSMT3 based expression vector. Enterokinase digestion of recombinant Nocardia GCH, and in-gel digestion of Nocardia GCH and recombinant GCH followed by MALDI-TOF-MS analysis, confirmed that the actual subunit size of the enzyme was 24.5kDa. Thus, we conclude that the active form of native Nocardia GCH is a decamer. Our earlier incorrect conclusion was that the native enzyme was an octamer derived from the anomalous SDS-PAGE migration of the subunit.
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Aimin
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Division of Medicinal and Natural Products Chemistry and Center for Biocatalysis and Bioprocessing, College of Pharmacy, University of Iowa, Iowa City, IA 42242, USA.
Simpson
D Randall
DR
Daniels
Lacy
L
Rosazza
John P N
JP
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006136:GTP Cyclohydrolase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009615:Nocardia / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D011994:Recombinant Proteins; D006136:GTP Cyclohydrolase