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J. Biol. Chem., Vol. 275, Issue 22, 16851-16856, June 2, 2000

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New -Lactamase Inhibitory Protein (BLIP-I) from Streptomyces exfoliatus SMF19 and Its Roles on the Morphological Differentiation^*

Sung Gyun Kang, Hyeon Ung Park, Hyun Sook Lee, Hyoung Tae Kim, and Kye Joon Lee

From the School of Biological Sciences, Seoul National University, Seoul 151-742, Korea

A new -lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 was purified and characterized. The molecular mass of BLIP-I was estimated to be 17.5 kDa by gel filtration fast protein liquid chromatography. The N-terminal sequence was NH₂-Asn-Ser-Gly-Phe-Ser-Ala-Glu-Lys-Tyr-Glu-Gln-Ile-Gln-Phe-Gly. BLIP-I inhibited Bacto® Penase (Difco), and plasmid encoded TEM-1 -lactamase, whereas it did not inhibit Enterobacter cloacae -lactamases. The K_i value of BLIP-I against TEM-1 -lactamase was determined to be 0.047 nM. The gene (bliA) encoding BLIP-I protein was identified by screening a genomic library using an oligonucleotide probe with a sequence based on the N-terminal sequence of BLIP-I. Analysis of the nucleotide sequence revealed that the gene was 558 base pairs in length and encoded a mature protein of 157 amino acid residues preceded by a 29-amino acid signal sequence. Pairwise comparison of the deduced amino acid sequence showed 38% identity with BLIP of Streptomyces clavuligerus. Furthermore, the 49th amino acid residue of BLIP-I was identical to Asp-49 of BLIP that was characterized to be an important residue for the inhibitory activity of BLIP. A modified BLIP-I in which Asp-49 was replaced by alanine (D49A) was obtained by site-directed mutagenesis. The inhibitory activities of recombinant (r) BLIP-I and its D49A mutant derivative, expressed in Escherichia coli, were compared. The K_i value of rBLIP-I against TEM-1 -lactamase was similar to that of wild-type BLIP-I, but the D49A mutation increased the K_i of rBLIP-I inhibition approximately 200-fold. A disruption mutant of the bliA gene in S. exfoliatus SMF19 was obtained by replacing the wild-type bliA gene with a copy inactivated by inserting a hygromycin resistance gene. The disruption mutant showed a bald phenotype, indicating that the bliA gene plays a role in morphological differentiation.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF201389.

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