Nucleotide sequence and expression of a Streptomyces griseosporeus proteinaceous alpha-amylase inhibitor (HaimII) gene

Abstract

The coding region of the alpha-amylase inhibitor (HaimIl) gene from the producing strain Streptomyces griseosporeus YM-25 was localized on an 800-base-pair DNA segment. The nucleotide sequence of a 1,191-base-pair region including the HaimIl gene was determined by the dideoxy-chain termination method. The nucleotide sequence data predicted an open reading frame of 363 base pairs starting with an ATG initiation codon and ending with a TGA translational stop codon. The amino acid sequence deduced from the nucleotide sequence indicated that the presumptive pre-HaimIl protein extends 37 amino acids to the amino terminus and 6 amino acids to the carboxyl terminus of the mature HaimIl protein. The pre-HaimIl protein is believed to be processed both during and after secretion. Two forms of the inhibitor, which have a higher molecular weight than that of the HaimIl protein isolated from S. griseosporeus, were partially purified from the culture filtrate of Streptomyces lividans containing the cloned HaimIl gene.Various proteinaceous alpha-amylase inhibitors from microorganisms have been isolated and characterized (3,7,9,20,33,34). These inhibitors are active against alpha-amylases of animal origin but inactive towards those of plants and microorganisms. Haim, a proteinaceous alpha-amylase inhibitor isolated from the culture filtrate of Streptomyces griseosporeus YM-25 (7, 21), also inhibits alpha-amylases of animal origin. Two isoinhibitors produced by S. griseosporeus, Haiml and Haimll, have similar inhibitory specificities and amino acid compositions but differ in their isoelectric points. The amino acid sequence of the Haimll protein has been determined by conventional sequencing methods (20) and found to bear considerable sequence homology with other alpha-amylase inhibitors from Streptomyces species, such as Hoe467A and A13688 (3, 9, 34). Furthermore, these proteins possess four cysteine residues at exactly identical positions and constitute two disulfide bonds, which make up the rigid and stable structure of the inhibitor molecules. Conceivably they have a similar configuration. The recent determination of the three-dimensional structure of the Hoe467A protein largely depended on the small and rigid structure of the molecules (14, 24). These characteristics of alpha-amylase inhibitors of Streptomyces origin appear to be amenable for the study of structure and function of protein molecules by protein engineering.The mechanism of alpha-amylase inhibition by HaimlI and the other inhibitors from Streptomyces spp. is attributed to specific binding of the inhibitor molecule to the active site of alpha-amylase (33). It is reasonable to believe that these alpha-amylase inhibitors will be useful for the study of specific protein-protein interactions.Previously we reported the successful cloning of the (25) is a composite plasmid consisting of pUC9 (37) and pIJ702 (13). YEME medium (10) was used for the cultivation of Streptomyces strains for protoplast transformation and HaimIl production. Streptomyces strains were cultiv...

Heterologous expression of the alpha-amylase inhibitor gene cloned from an amplified genomic sequence of Streptomyces tendae

“…There is clearly synthesis and secretion of tendamistat in yields similar to those from pIJ702, from which vector pIJ486 was derived (Fig. 2B) (32 (24). Boxed in the nucleotide sequences are putative ribosomal-binding sites.…”

“…Additionally, we show that the nucleotide sequence and the processing of tendamistat are significantly different to those of Haim II of Streptomyces griseosporus, whose primary and secondary protein structures are similar to those of tendamistat (1,24).…”

Molecular Cloning and Expression of Proteinaceousα-Amylase Inhibitor Gene fromStreptomyces nitrosporeusinEscherichia coli

“…These inhibitor-amylase interactions will offer a model for the study of specific protein-protein interactions. Our previous study about chemical modification of Haim showed that the arginine residue (equivalent to Arg (17) in T-76 inhibitor; Fig. 5) was necessary to express inhibitory activity.35) It seems reasonable to assume that this arginine residue might contribute towards binding to a target site on the a-amylase molecule because it occurs in the portion highly conserved among proteinaceous a-amylase inhibitors, while the specificity of the inhibitor towards a-amylases should be attributed to amino acid residues in the carboxy-terminal half, which are not so highly conserved.…”

“…The isolation and some properties of T -76 inhibitor were described in our previous report. 6) The structural genes of Haim 16,17) and Tendamistat 1S ) have been cloned and characterized. It is important to clone the other inhibitor genes and make their primary structures clear so as to obtain more information on the relationship between inhibitory specificity and amino acid sequence.…”

Heterologous expression and secretion of a Streptomyces scabies esterase in Streptomyces lividans and Escherichia coli

“…lividans, the gene products are typically secreted (5,8,12,15,25,29,34). In some cases, the site for signal sequence processing in S. lividans was demonstrated to be identical to that for processing in the native host strain (8,25).…”