Abstract
The flavin adenine dinucleotide (FAD)-containing putrescine oxidase of Micrococcus rubens catalyses the oxidative deamination of putrescine. The amino acid sequences of the NH2-termini of the mature enzyme and lysyl-endopeptidase-generated fragments were determined for preparation of synthetic oligonucleotides as hybridization probes for cloning. A 4.4 kb BamHI fragment which contained DNA sequences hybridizing to the probes was cloned in pUC19 in Escherichia coli. The nucleotide sequence together with the determined amino acid sequences revealed that this enzyme consists of 480 amino acids (M(r) 52,000) and contains an FAD-binding consensus sequence at its NH2-terminal portion. In front of the transcriptional start point, which is 28 bases upstream of the initiation codon as determined by primer extension, -35 and -10 sequences similar to typical prokaryotic promoter consensus sequences are present. E. coli JM109 containing the putrescine oxidase gene just downstream of the lac promoter in pUC18 produced a large amount of this protein when grown at 37 degrees C but in the enzymically inactive form of inclusion bodies. However, cultivation of the recombinant E. coli cells at temperatures below 30 degrees C led to production of active enzyme (20 times as much as produced by the original M. rubens strain).
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