Isolation, sequence and overexpression of the gene encoding NAD-dependent formate dehydrogenase from the methylotrophic yeast Candida methylica.
NAD-dependent formate dehydrogenase (FDH) was isolated from Candida methylica (Cm) grown on 0.5% methanol. Its N-terminal amino acid (aa) sequence was determined, as was that of a commercial FDH from Candida boidinii. Degenerate oligodeoxyribonucleotides were made to the 5' region of the fdh gene from Cm using this information and to the 3' region using C-terminal aa sequence data from the methylotropic yeast, Hansenula polymorpha. An almost complete 1.1-kb fragment was amplified from Cm genomic DNA via the polymerase chain reaction (PCR). This fragment was cloned, sequenced and used to probe a Southern blot, from which a 3.4-kb EcoRI fragment containing the fdh open reading frame (ORF) was isolated. The complete nucleotide sequence of this fdh ORF was determined and corresponds to a protein of 364 aa (40,343 Da). The ORF of fdh was cloned into pKK223-3 using PCR and transformed into Escherichia coli. Enzymatically active FDH was produced to 15% of soluble E. coli protein. The deduced aa sequence of this FDH is compared to the aa sequences of four known FDH, from bacteria, yeast, fungi and plant mitochondria.
Allen
S J
SJ
Molecular Recognition Centre, School of Medical Sciences, University of Bristol, UK.
Holbrook
J J
JJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D015139:Blotting, Southern; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005560:Formate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics*; D005800:Genes, Fungal; D000432:Methanol / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000596:Amino Acids; D011994:Recombinant Proteins; D005560:Formate Dehydrogenases; D000432:Methanol