Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.

A second open reading frame, fkbD, was found upstream of fkbM in all three aforementioned species and was predicted to encode a protein of 388 residues that showed a strong resemblance to cytochrome P-450 hydroxylases. Disruption of fkbD had a polar effect on the synthesis of the downstream fkbM gene product and resulted in the formation of 9-deoxo-31-O-demethyl-FK506. This established the product of fkbD as the cytochrome P-450 9-deoxo-FK506 hydroxylase, which is responsible for hydroxylation at position C-9 of the FK506 and FK520 macrolactone ring.The polyketide, immunosuppressant compound FK506 ( Fig. 1) (13) is a 23-membered macrolide with potent antifungal activity produced by several Streptomyces species. FK506 is approximately 100-fold more potent than the structurally unrelated immunosuppressive compound cyclosporin A. Both drugs are important therapeutic agents for the prevention of graft rejection following organ and bone marrow transplantations and for the treatment of autoimmune diseases (22). FK520 (also known as immunomycin and ascomycin) is another immunosuppressive compound similar to FK506 (Fig. 1) in which the allyl group is replaced by an ethyl group at position C-21 of the macrolactone ring (9). Both the antifungal and the immunosuppressive activities of FK520 are approximately one-half of those exhibited by FK506 (9).Through precursor incorporation experiments, Byrne et al. (3) demonstrated that the polyketide portion of FK506 and FK520 is derived, for the most part, from acetate and propionate. Those authors also established the origin of the pipecolate and the cyclohexyl rings to be lysine and shikimic acid, respectively, and demonstrated that the source of the methyl portion of the methoxyl groups at C-13, C-15, and C-31 of FK520 (Fig. 1) is L-methionine.The enzymology of FK506 biosynthesis has also been explored to some extent. The pipecolate-activating enzyme which presumably incorporates pipecolate into the completed polyketide chain has been characterized previously (19). Both 31-O-demethyl-FK520 methyltransferase and 31-O-demethyl-FK506 methyltransferase (FKMT) have been isolated from the producing strains (3, 27). These two enzymes can use each other's substrate interchangeably and methylate the C-31 OH and not the C-13 or C-15 OH group (27).Here, we report the isolation and molecular characterization of two genes involved in the biosynthesis of FK506. One gene, fkbM, encodes FKMT, and the other, fkbD, encodes a cytochrome P-450 9-deoxo-FK506 hydroxylase that catalyzes hydroxylation at C-9. MATERIALS AND METHODSStandard recombinant DNA techniques were performed as described by Sambrook et al. (24).Probe design. N-terminal amino acid sequencing of FKMT from Streptomyces sp. strain MA6858 (27) gave a 39-mer with the sequence SDVVETLRLPNGA TVAHVNAGEAQFLYREIFTDRCYLRH. This peptide sequence was then used to design two nonoverlapping degenerate oligonucleotide probes, P1 and P2, in which inosine was incorporated at the third position of highly degenerate codons (2). P1 corresponded to ...

The immunosuppressant FK506 is a 23-membered macrocyclic polyketide produced by several Streptomyces species. Sequencing of a 19.5-kb contiguous segment of DNA from the FK506 gene cluster of Streptomyces sp. MA6548 revealed the presence of a single 19.3-kb open reading frame designatedfkbA. fkbA encodes a component of the FK506 polyketide synthase, a complex enzyme system which catalyzes synthesis of the polyketide portion of FK506. The predicted product of genefkbA is a 630660-Da protein (6420 amino acids) that contains 1 9 independent domains with a high degree of amino acid sequence similarity to the catalytic activities of known fatty acid synthases. The identified domains are arranged into four repeated modules with a linear organization precisely as that of animal fatty acid synthase and type I polyketide synthase. Each module participates in one round of chain extension and subsequent processing and thus FkbA polypeptide catalyzes four of the ten condensation steps required for synthesis of the FK506 macrolactone ring. Disruption of jkbA results in the generation of an FK506 non-producing mutant demonstrating direct involvement of fkbA in the biosynthesis of FK506.

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EnvZ and OmpR are a transmembrane sensor and its cognate response regulator, respectively, regulating the transcription of porin genes in response to medium osmolarity in Escherichia coli. The cytoplasmic domain of EnvZ (EnvZc) possesses both kinase and phosphatase activities and can be dissected into two functional domains, A and B. Here, we performed a cysteine-scanning analysis of domain A, a 67-residue central dimerization and phosphatase domain containing His-243 as the phosphorylation site, and we examined the effects of the cysteine substitution mutations on the enzymatic activities of domain A. The substitution mutations were made at 31 residues, from which 24 mutant domain A proteins were biochemically characterized. From the analysis of the phosphatase activity of purified mutant proteins, it was found that there are two regions in domain A which are important for this activity. Cysteine mutations in these regions dramatically reduce or completely abolish the phosphatase activity of domain A. The mutations that have the most-severe effects on domain A phosphatase activity also significantly reduce the phosphatase activity of EnvZc containing the same mutation. Using an in vitro complementation system with EnvZc(H243V), these cysteine mutants were further characterized for their autophosphorylation activities as well as their phosphotransfer activities. The results indicate that some mutations are specific either for the phosphatase activity or for the kinase activity.