Two proteins that act as alpha-amylase inhibitors, Haim I and Paim I, were crystallized and preliminary X-ray diffraction studies on them were carried out. We also sequenced Haim I prepared from Streptomyces griseosporeus YM-25 and confirmed that it is composed of 78 amino acid residues. Crystals of Haim I were grown from ammonium sulfate solution mixed with ethanol by the vapor diffusion technique. The crystals grew as hexagonal bipyramids and diffracted X-rays beyond 2.0 A resolution. They belong to the space group P6(1)22 (or P6(5)22) with unit cell dimensions of a = b = 36.7 A, c = 192.4 A, and contain one molecule per asymmetric unit. Paim I, a protein of 39 amino acid residues produced by Streptomyces corchorusii, was crystallized under similar conditions to Haim I. The crystals diffracted X-rays beyond 2.5 A. They belong to the space group P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = b = 65.4 A, c = 96.1 A, and contain three molecules per asymmetric unit.

The coding region of the alpha-amylase inhibitor (HaimII) 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 HaimII 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-HaimII protein extends 37 amino acids to the amino terminus and 6 amino acids to the carboxyl terminus of the mature HaimII protein. The pre-HaimII 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 HaimII protein isolated from S. griseosporeus, were partially purified from the culture filtrate of Streptomyces lividans containing the cloned HaimII gene.

The amino acid sequence of a protein alpha-amylase inhibitor from Streptomyces griseosporeus YM-25 (Haim II), which consists of 77 amino acid residues, including two disulfide bridges, was determined by conventional methods. One of the disulfide bridges was found to be located between Cys(6) and Cys(22), and the other between Cys(40) and Cys(67) from the results of structure analyses of the two cystine-containing peptides obtained from the thermolysin digest of the native inhibitor.