In our studies of structure-function correlation of polypeptides we used Tendamistat (TM), an alpha-amylase-inhibitor of Streptomyces tendae, as a model to investigate the influence of different mutants on the expression and secretion of the protein. In addition, we examined the influence of replacing the two disulphide-bridges that stabilize the two-loop structure of the whole protein. The single mutants C27S, C27T, C45A, the double mutants C11A/C27A, C11A/C27S, C11A/C27T, C11A/C27L, C45/C73A and the fourfold mutant C11A/C27A/C45A/C73A were prepared. The mutated TM gene was expressed in S. lividans TK 24, which secretes the active form of the inhibitor into the culture medium. Compared with the wild-type, the double-mutated TM derivatives show an increase in secreted protein by a factor of two to ten. In contrast, the single-mutated inhibitor analogues show the reverse effect. In order to examine the influence of temperature and culture media on the production of protein derivative we used the most unstable C11A analogue. Our expression studies at 10, 19, 28 and 37 degrees C established 19 degrees C as the optimal temperature for production of the protein derivatives. The correlation between the stability and secretion of TM is discussed in the context of our knowledge of protein translocation in bacteria. Based on these experiments we optimized the fermentation parameters, isolated TM analogous on a large scale, and verified them.

The coding region for a secreted proteinaceous inhibitor of the human a-amylase (tendamistat; HOE 467) was identified by using a synthetic oligonucleotide probe. Inhibitors of the human a-amylase are of high potential pharmaceutical value as regulative agents in reducing digestive-starch degradation in patients suffering from diabetes and obesity (29). Proteins inhibiting human salivary gland amylases were isolated from plants (7,22) and various microorganisms (1, 9, 23). The ac-amylase inhibitor of Streptomyces tendae is a biochemically well-characterized acidic protein of 74 amino acids (30) secreted in large amounts into the culture medium (15). Recently, the complete threedimensional structure has been elucidated by X-ray crystallography (26) and independently by nuclear magnetic resonance spectroscopy (14). Tendamistat specifically inhibits mammalian oa-amylases (30) and also some Streptomyces amylases (17). An outstanding feature of tendamistat is the almost irreversible binding to the human ax-amylase (30). However, how the inhibitor binds to the enzyme and why the binding is irreversible are unknown.To apply protein-engineering techniques to understand the mode of action of the inhibitor and to study the mechanism of protein secretion in Streptomyces spp., a group of industrially familiar, filamentous, gram-positive microorganisms, we had to clone the inhibitor gene.In the course of a strain improvement program, an overexpressing mutant of S. tendae which is characterized by amplified sequences in its genome was isolated (15). Hybridization experiments using a synthetic oligonucleotide probe suggested the presence of the a-amylase inhibitor gene on the amplified sequence (16).In the present paper we describe the Assay of inhibitor production. Selection of recombinant strains secreting the inhibitor was carried out on agar plates as described elsewhere (16). Synthesis of tendamistat in liquid cultures was measured by using an automated version of the ferricyanide reducing sugar analysis (2). For polyacrylamide gel electrophoresis, samples of culture filtrates were used without further concentration. Electrophoretic mobilities of the secreted proteins were analyzed on native 15% polyacrylamide gels by using the insulin la system (20). Gels were stained with Coomassie blue R250.

The recombinant mutant a-amylase inhibitor [R19L]Tendamistat, with Argl9 replaced by Leu, was prepared and its NMR solution structure determined. Based on complete sequence-specific 'H-NMR assignments, 845 nuclear Overhauser effect upper-distance constraints and 156 dihedral angle constraints were collected using two-dimensional homonuclear 'H-NMR experiments. The structure was calculated with the program DIANA, using the redundant dihedral angle constraints strategy for improved convergence. For restrained energy minimization, the programs FANTOM and AM-BER were used. The wild-type NMR solution structure was similarly recalculated from the previously published input of conformational constraints [Kline, A., Braun, W. & Wuthrich, K. (1988) J. Mol. Biol. 204,. For each protein a group of 20 conformers represents a well-defined solution structure, with average root-mean-square-distance values for the backbone atoms of the individual conformers relative to the mean coordinates of 50 pm. The two structures are nearly identical to each other and to the previously published Tendamistat structures in solution and in crystals. The only significant difference is strictly localized near the single amino acid substitution in the presumed active site -Trpl8-Arg(Leu)-Tyr-, i.e. Leu19 and Tyr2O are more precisely defined in the solution structure of IR19LlTendamistat than the corresponding residues Argl9 and Tyr2O in wild-type Tendamistat.Tendamistat is an a-amylase inhibitor from Streptomyces tendue consisting of a 74-amino-acid polypeptide [ 11, which has played a key role in the development of three-dimensional protein-structure determination by NMR spectroscopy in solution [2]. In fact, although the first NMR structure determinations were those of glucagon [3, 41 and protease inhibitor IIA from bull seminal plasma [5], Tendamistat has been considered by many to be the first true high-resolution

The Streptomyces-amylase inhibitor tendamistat is secreted by a signal peptide with an amino-terminal charge of 3. To elucidate the influence of the charged residues on protein secretion in Streptomyces, the ami-no-terminal charge was varied from 6 to neutral net charge. The effects of charge variation were analyzed in combination with three Streptomyces promoters and two transcriptional terminators. Introduction of additional positive charges significantly decreased the amount of secreted tendamistat. On the contrary, a charge reduction to 2 resulted in the doubling of in-hibitor production. After exclusion of transcriptional effects, the observed alterations of inhibitor secretion by the mutants with a charge of 6 to 2 were attributed to a modulation of precursor synthesis. Furthermore , a tight coupling of synthesis and export was stated. Charge reduction to 1 or neutral charge generally reduced the yield of secreted tendamistat, yet remarkable differences were found for mutants with identical net charge. Elimination of the positive charge at a defined position resulted in the release of tendamistat precursor protein, which suggested a specific uncou-pling of synthesis and translocation.