The activation of protein kinase B/Akt is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. Because insulin-like growth factor 1 stimulates the resumption of meiosis in Xenopus laevis oocytes via phosphoinositide 3-kinase activation, we investigated the Akt involvement in this process. Injection of mRNA coding for a constitutively active Akt in Xenopus oocytes induced germinal vesicle breakdown (GVBD) to the same extent as progesterone or insulin treatment. Injection of mRNA coding for the wild type Akt kinase was less effective in stimulating GVBD, whereas Akt bearing a lysine mutation in the catalytic domain that abolishes the kinase activity had no effect. A mutant Akt lacking a membrane-targeting sequence did not induce GVBD, despite high levels of expression and activity. As previously reported for insulin, induction of GVBD by Akt was prevented by incubating the oocytes with cilostamide, an inhibitor specific for the type 3 phosphodiesterase (PDE3), suggesting that the activity of a PDE is required for Akt action. That an increase in PDE activity in the oocyte is sufficient to induce meiotic resumption was demonstrated by expression of an active PDE protein. In addition, the constitutively active Akt caused a 2-fold increase in the activity of the endogenous PDE. These data demonstrate that Akt is in the pathway controlling resumption of meiosis in theXenopus oocyte and that regulation of the activity of a PDE3 is a step distal to the kinase activation. The activation of protein kinase B/Akt is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. Because insulin-like growth factor 1 stimulates the resumption of meiosis in Xenopus laevis oocytes via phosphoinositide 3-kinase activation, we investigated the Akt involvement in this process. Injection of mRNA coding for a constitutively active Akt in Xenopus oocytes induced germinal vesicle breakdown (GVBD) to the same extent as progesterone or insulin treatment. Injection of mRNA coding for the wild type Akt kinase was less effective in stimulating GVBD, whereas Akt bearing a lysine mutation in the catalytic domain that abolishes the kinase activity had no effect. A mutant Akt lacking a membrane-targeting sequence did not induce GVBD, despite high levels of expression and activity. As previously reported for insulin, induction of GVBD by Akt was prevented by incubating the oocytes with cilostamide, an inhibitor specific for the type 3 phosphodiesterase (PDE3), suggesting that the activity of a PDE is required for Akt action. That an increase in PDE activity in the oocyte is sufficient to induce meiotic resumption was demonstrated by expression of an active PDE protein. In addition, the constitutively active Akt caused a 2-fold increase in the activity of the endogenous PDE. These data demonstrate that Akt is in the pathway controlling resumption of meiosis in theXenopus oocyte and that regulation of the activity of a PDE3 is a step distal to the kinase activation. A critical step in growth factor stimulation of the target cell is the activation of the phosphoinositide 3-kinase (PI3-K) 1The abbreviations used are: PI3-K, phosphatidylinositol 3-kinase; PDE, phosphodiesterase; IGF-1, insulin-like growth factor 1; HA, hemagglutinin; myr, myristoylation; PH, pleckstrin homology; GVBD, germinal vesicle breakdown; WT, wild type. 1The abbreviations used are: PI3-K, phosphatidylinositol 3-kinase; PDE, phosphodiesterase; IGF-1, insulin-like growth factor 1; HA, hemagglutinin; myr, myristoylation; PH, pleckstrin homology; GVBD, germinal vesicle breakdown; WT, wild type. pathway. This signaling pathway has been implicated in a wide array of cellular events including mitogenesis, transformation, differentiation, and regulation of metabolism (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar). Recently Akt, also known as protein kinase B or related to the A and protein kinase C (2Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (387) Google Scholar, 3Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (439) Google Scholar, 4Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (786) Google Scholar), was identified as a kinase distal to PI3-K (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar). Three Akt isoforms (α, β, and γ) with closely related properties have been identified. These are proteins of approximately 60 kDa containing a pleckstrin homology (PH) domain (5Haslam R.J. Koide H.B. Hemmings B.A. Nature. 1993; 363: 309-310Crossref PubMed Scopus (386) Google Scholar) and a serine/threonine kinase domain structurally related to the catalytic domains of protein kinases A and C. Phosphatidylinositol 3,4-bisphosphate and 3,4,5-trisphosphate, the products of PI3-K, bind to the PH domain of Akt and serve to anchor the enzyme to the membrane as well as to induce a conformational change in the enzyme (6Hemmings B.A. Science. 1997; 277: 534Crossref PubMed Scopus (77) Google Scholar). The subsequent phosphorylation of Akt, at Thr-308 in the catalytic domain and Ser-473 of the C terminus of Aktα, is crucial for the activation of Akt (7Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 8Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar). At least one of the kinases phosphorylating Akt has been identified as the 3-phosphoinositide-dependent kinase-1 (9Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 10Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 11Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1045) Google Scholar).Akt is activated by insulin and IGF-1 (12Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar), and this regulation mediates the activation of glucose uptake (13Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar), the phosphorylation and deactivation of GSK3 (14Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar), and activation of p70 S6 kinases (15Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar). Akt also phosphorylates the proapoptotic protein Bad, promoting its interaction with 14-3-3 and thereby preventing apoptosis (16Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar).In somatic cells including adipocytes, insulin regulates the activation of cGMP-inhibited phosphodiesterase (PDE3) (17Degerman E. Smith C.J. Tornqvist H. Vasta V. Belfrage P. Manganiello V.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 533-537Crossref PubMed Scopus (164) Google Scholar). This activation is mediated by PI3-K because wortmannin, a PI3-K inhibitor, blocks the insulin-dependent activation of the PDE (18Rahn T. Ridderstrale M. Tornqvist H. Manganiello V. Fredrikson G. Belfrage P. Degerman E. FEBS Lett. 1994; 350: 314-318Crossref PubMed Scopus (119) Google Scholar). The PDE3B isoform expressed in adipocytes is phosphorylated following insulin stimulation, and the phosphorylation is associated with an increase in PDE activity (19Rahn T. Ronnstrand L. Leroy M.J. Wernstedt C. Tornqvist H. Manganiello V.C. Belfrage P. Degerman E. J. Biol. Chem. 1996; 271: 11575-11580Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This PDE activation and the consequent decrease in intracellular cAMP are probably responsible for the decrease in the hormone-sensitive lipase activity (17Degerman E. Smith C.J. Tornqvist H. Vasta V. Belfrage P. Manganiello V.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 533-537Crossref PubMed Scopus (164) Google Scholar) and for the antilipolytic effects of insulin. The properties of the kinase phosphorylating PDE3B have not been firmly established, although a recent report indicates that Akt coelutes with a kinase activity that phosphorylates PDE3B in a cell-free system (20Wijkander J. Landstrom T.R. Manganiello V. Belfrage P. Degerman E. Endocrinology. 1998; 139: 219-227Crossref PubMed Scopus (114) Google Scholar).In Xenopus laevis oocytes, IGF-1 induces the dissolution of the nuclear membrane (GVBD) and completion of the first meiotic division (21Maller J.L. J. Cyclic Nucleotide Protein Phosphor. Res. 1986; 11: 543-555PubMed Google Scholar). Although distinct signaling pathways may be activated, IGF-1, or the physiological stimulus progesterone, activates meiotic resumption by inducing a transient decrease in intraoocyte cAMP levels, thereby reducing the protein kinase A activity (22Maller J.L. Butcher F.R. Krebs E.G. J. Biol. Chem. 1979; 254: 579-582Abstract Full Text PDF PubMed Google Scholar, 23Sadler S.E. Maller J.L. J. Biol. Chem. 1987; 262: 10644-10650Abstract Full Text PDF PubMed Google Scholar). It has been proposed that activation of a PDE and a decrease in cAMP are crucial steps for reentry into the cell cycle. The effects of insulin and IGF-1 on meiosis are blocked by cilostamide, a specific inhibitor of the type 3 family of phosphodiesterases (24Sadler S.E. Mol. Endocrinol. 1991; 5: 1939-1946Crossref PubMed Scopus (42) Google Scholar). Furthermore, insulin treatment or injection of an activated Ras leads to an increase in the PDE activity in the Xenopus oocyte (25Sadler S.E. Maller J.L. J. Biol. Chem. 1989; 264: 856-861Abstract Full Text PDF PubMed Google Scholar).Here we have tested the hypothesis that Akt is involved in the IGF-1-induced resumption of meiosis by activating a PDE present in theXenopus oocyte. Our data demonstrate that Akt is a signal for resumption of meiosis in the Xenopus oocyte and that activation of a PDE3 is a step distal to the Akt activation.DISCUSSIONAkt is a serine-threonine kinase in the PI3-K signaling pathway that mediates growth factor regulation of cell differentiation and survival (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar). Here we provide evidence for an additional role of this Akt kinase in the control of the cell cycle and meiosis. Akt expression in the Xenopus oocyte causes germinal vesicle breakdown and mitogen-activated protein kinase phosphorylation, hallmarks of the resumption of meiosis. Furthermore, our findings indicate that a PDE3 is distal to Akt activation. This conclusion is supported by both the pharmacological manipulation of the oocyte PDE and by the observation that an increase in PDE activity follows expression of the Akt kinase in oocyte. Finally, the finding that expression of a PDE in the oocyte per se is sufficient to promote a meiotic resumption is consistent with the above conclusions.Numerous observations have underscored the importance of the lipid PI3-K in insulin and growth factor signaling in somatic cells (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar) as well as in Xenopus oocytes. Because insulin and IGF-1 effects on meiosis are inhibited by wortmannin (29Liu X.J. Sorisky A. Zhu L. Pawson T. Mol. Cell. Biol. 1995; 15: 3563-3570Crossref PubMed Scopus (70) Google Scholar), PI3-K has been implicated in oocyte maturation. Injection of a constitutively active PI3-K mRNA causes resumption of meiosis (30Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (516) Google Scholar), whereas injection of the lipid phosphatase SIP/SHIP blocks the insulin effects (31Deuter-Reinhard M. Apell G. Pot D. Klippel A. Williams L.T. Kavanaugh W.M. Mol. Cell. Biol. 1997; 17: 2559-2565Crossref PubMed Scopus (60) Google Scholar). Our data extend these observations by identifying downstream steps in the pathway by which IGF-1 induces resumption of meiosis. As for somatic cells where the PI3-K effects are mediated by activation of Akt, we have shown that Akt activates resumption of meiosis in a manner similar to insulin/IGF-1.Although the myr-Akt efficiently promoted oocyte maturation, a wild type Akt was only partially effective in the oocyte model, consistent with observations in mammalian cells showing that wild type Akt has low basal activity in the absence of insulin or growth factor signals (7Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar,8Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 12Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 13Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar). Unlike the myr-Akt, the A2myr-Akt lacking the PH domain and with a mutation in the myristoylation signal was ineffective in inducing resumption of meiosis. This protein was efficiently expressed, and the overall Akt activity recovered in oocyte extracts was comparable with that obtained by expression of myr-Akt. When the activity of A2myr-Akt was corrected for the amount of protein expressed, this mutant kinase had one-fourth the specific activity of the myr-Akt. Because the only difference between A2myr-Akt and myr-Akt is the ability to interact with the lipid bilayer, we can conclude that Akt interaction with lipids is essential for the signaling meiotic resumption.Our findings that PDE3 inhibitors block the oocyte maturation induced by insulin and Akt, but not progesterone, indicate that a PDE3 is involved in the PI3-K signal transduction pathway activated by IGF-1/insulin. In agreement with this hypothesis, we have shown that insulin, PI3-K, and Akt stimulate the activity of PDE3 endogenous to the oocyte. That Akt activation of PDE3 may be a sufficient signal for meiosis resumption is strongly suggested by the observation that expression of a PDE causes GVBD. Our conclusion is consistent with the finding in adipocytes, where it has been shown that insulin through the PI3-K pathway activates PDE3B (32Degerman E. Belfrage P. Manganiello V.C. Biochem. Soc. Trans. 1996; 24: 1010-1014Crossref PubMed Scopus (27) Google Scholar).Our data do not exclude the possibility that Akt is at a branch point in the signaling pathway controlling maturation. Upon activation, this kinase may phosphorylate several substrates in addition to a PDE3, and the activation of a PDE3 has only a permissive role on meiotic maturation. According to this model, both the release of a cAMP-dependent blockade and the activation of a distinct signaling cascade may be required for meiotic resumption. For instance, Akt activates the p70 ribosomal S6 kinase, which may be involved in the regulation of mos translation, a crucial step in oocyte meiosis (33Sagata N. Oskarsson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-525Crossref PubMed Scopus (462) Google Scholar).Regardless of the mechanism of Akt action, our findings indicate a role for this enzyme in oocyte maturation. Interestingly, the cloning of 3-phosphoinositide-dependent kinase-1, the kinase-phosphorylating Thr-308 of Aktα, has uncovered a structural and function homology with the Drosophila DSTPK61 kinase. Although little is known about the function of the PI3-K and Akt pathway in Drosophila, DSTPK61 kinase may play an important role in the differentiation of the female and male germ cells (9Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). Our data in the Xenopus oocyte are in line with the view that this pathway is involved in oocyte maturation. The physiological signals activating the PI3-K and the Akt pathway are unknown at present but may be a means by which somatic cells control the function of germ cells. It is worth noting that the PDE3A mRNA (34Tsafriri A. Chun S.Y. Zhang R. Hsueh A.J. Conti M. Dev. Biol. 1996; 178: 393-402Crossref PubMed Scopus (266) Google Scholar) and protein are expressed in mammalian oocytes. Consistent with that shown inXenopus, PDE3 inhibitors block the spontaneous resumption of meiosis in rat and mouse oocytes as well as the maturation induced by the luteinizing hormone in the intact follicle (34Tsafriri A. Chun S.Y. Zhang R. Hsueh A.J. Conti M. Dev. Biol. 1996; 178: 393-402Crossref PubMed Scopus (266) Google Scholar, 35Wiersma A. Hirsh B. Tsafriri A. Hanssen R.J.G.M. Van de Kant M. Kloosterboer H.J. Conti M. Hseuh A.J.W. XIth International Workshop on Development and Function of the Reproductive Organs. 1998; (Amsterdam, Netherlands): 15Google Scholar). Recentin vivo observations have further confirmed the crucial role of PDE3 for resumption of meiosis in mammals (35Wiersma A. Hirsh B. Tsafriri A. Hanssen R.J.G.M. Van de Kant M. Kloosterboer H.J. Conti M. Hseuh A.J.W. XIth International Workshop on Development and Function of the Reproductive Organs. 1998; (Amsterdam, Netherlands): 15Google Scholar). Thus, our data on the Xenopus oocyte open the possibility that, in the mammalian follicle, physiological signals promoting resumption of meiosis regulate the intraoocyte cAMP levels via the PI3-K/Akt signaling pathway. A critical step in growth factor stimulation of the target cell is the activation of the phosphoinositide 3-kinase (PI3-K) 1The abbreviations used are: PI3-K, phosphatidylinositol 3-kinase; PDE, phosphodiesterase; IGF-1, insulin-like growth factor 1; HA, hemagglutinin; myr, myristoylation; PH, pleckstrin homology; GVBD, germinal vesicle breakdown; WT, wild type. 1The abbreviations used are: PI3-K, phosphatidylinositol 3-kinase; PDE, phosphodiesterase; IGF-1, insulin-like growth factor 1; HA, hemagglutinin; myr, myristoylation; PH, pleckstrin homology; GVBD, germinal vesicle breakdown; WT, wild type. pathway. This signaling pathway has been implicated in a wide array of cellular events including mitogenesis, transformation, differentiation, and regulation of metabolism (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar). Recently Akt, also known as protein kinase B or related to the A and protein kinase C (2Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (387) Google Scholar, 3Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (439) Google Scholar, 4Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (786) Google Scholar), was identified as a kinase distal to PI3-K (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar). Three Akt isoforms (α, β, and γ) with closely related properties have been identified. These are proteins of approximately 60 kDa containing a pleckstrin homology (PH) domain (5Haslam R.J. Koide H.B. Hemmings B.A. Nature. 1993; 363: 309-310Crossref PubMed Scopus (386) Google Scholar) and a serine/threonine kinase domain structurally related to the catalytic domains of protein kinases A and C. Phosphatidylinositol 3,4-bisphosphate and 3,4,5-trisphosphate, the products of PI3-K, bind to the PH domain of Akt and serve to anchor the enzyme to the membrane as well as to induce a conformational change in the enzyme (6Hemmings B.A. Science. 1997; 277: 534Crossref PubMed Scopus (77) Google Scholar). The subsequent phosphorylation of Akt, at Thr-308 in the catalytic domain and Ser-473 of the C terminus of Aktα, is crucial for the activation of Akt (7Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 8Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar). At least one of the kinases phosphorylating Akt has been identified as the 3-phosphoinositide-dependent kinase-1 (9Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 10Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 11Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1045) Google Scholar). Akt is activated by insulin and IGF-1 (12Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar), and this regulation mediates the activation of glucose uptake (13Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar), the phosphorylation and deactivation of GSK3 (14Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar), and activation of p70 S6 kinases (15Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar). Akt also phosphorylates the proapoptotic protein Bad, promoting its interaction with 14-3-3 and thereby preventing apoptosis (16Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar). In somatic cells including adipocytes, insulin regulates the activation of cGMP-inhibited phosphodiesterase (PDE3) (17Degerman E. Smith C.J. Tornqvist H. Vasta V. Belfrage P. Manganiello V.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 533-537Crossref PubMed Scopus (164) Google Scholar). This activation is mediated by PI3-K because wortmannin, a PI3-K inhibitor, blocks the insulin-dependent activation of the PDE (18Rahn T. Ridderstrale M. Tornqvist H. Manganiello V. Fredrikson G. Belfrage P. Degerman E. FEBS Lett. 1994; 350: 314-318Crossref PubMed Scopus (119) Google Scholar). The PDE3B isoform expressed in adipocytes is phosphorylated following insulin stimulation, and the phosphorylation is associated with an increase in PDE activity (19Rahn T. Ronnstrand L. Leroy M.J. Wernstedt C. Tornqvist H. Manganiello V.C. Belfrage P. Degerman E. J. Biol. Chem. 1996; 271: 11575-11580Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This PDE activation and the consequent decrease in intracellular cAMP are probably responsible for the decrease in the hormone-sensitive lipase activity (17Degerman E. Smith C.J. Tornqvist H. Vasta V. Belfrage P. Manganiello V.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 533-537Crossref PubMed Scopus (164) Google Scholar) and for the antilipolytic effects of insulin. The properties of the kinase phosphorylating PDE3B have not been firmly established, although a recent report indicates that Akt coelutes with a kinase activity that phosphorylates PDE3B in a cell-free system (20Wijkander J. Landstrom T.R. Manganiello V. Belfrage P. Degerman E. Endocrinology. 1998; 139: 219-227Crossref PubMed Scopus (114) Google Scholar). In Xenopus laevis oocytes, IGF-1 induces the dissolution of the nuclear membrane (GVBD) and completion of the first meiotic division (21Maller J.L. J. Cyclic Nucleotide Protein Phosphor. Res. 1986; 11: 543-555PubMed Google Scholar). Although distinct signaling pathways may be activated, IGF-1, or the physiological stimulus progesterone, activates meiotic resumption by inducing a transient decrease in intraoocyte cAMP levels, thereby reducing the protein kinase A activity (22Maller J.L. Butcher F.R. Krebs E.G. J. Biol. Chem. 1979; 254: 579-582Abstract Full Text PDF PubMed Google Scholar, 23Sadler S.E. Maller J.L. J. Biol. Chem. 1987; 262: 10644-10650Abstract Full Text PDF PubMed Google Scholar). It has been proposed that activation of a PDE and a decrease in cAMP are crucial steps for reentry into the cell cycle. The effects of insulin and IGF-1 on meiosis are blocked by cilostamide, a specific inhibitor of the type 3 family of phosphodiesterases (24Sadler S.E. Mol. Endocrinol. 1991; 5: 1939-1946Crossref PubMed Scopus (42) Google Scholar). Furthermore, insulin treatment or injection of an activated Ras leads to an increase in the PDE activity in the Xenopus oocyte (25Sadler S.E. Maller J.L. J. Biol. Chem. 1989; 264: 856-861Abstract Full Text PDF PubMed Google Scholar). Here we have tested the hypothesis that Akt is involved in the IGF-1-induced resumption of meiosis by activating a PDE present in theXenopus oocyte. Our data demonstrate that Akt is a signal for resumption of meiosis in the Xenopus oocyte and that activation of a PDE3 is a step distal to the Akt activation. DISCUSSIONAkt is a serine-threonine kinase in the PI3-K signaling pathway that mediates growth factor regulation of cell differentiation and survival (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar). Here we provide evidence for an additional role of this Akt kinase in the control of the cell cycle and meiosis. Akt expression in the Xenopus oocyte causes germinal vesicle breakdown and mitogen-activated protein kinase phosphorylation, hallmarks of the resumption of meiosis. Furthermore, our findings indicate that a PDE3 is distal to Akt activation. This conclusion is supported by both the pharmacological manipulation of the oocyte PDE and by the observation that an increase in PDE activity follows expression of the Akt kinase in oocyte. Finally, the finding that expression of a PDE in the oocyte per se is sufficient to promote a meiotic resumption is consistent with the above conclusions.Numerous observations have underscored the importance of the lipid PI3-K in insulin and growth factor signaling in somatic cells (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar) as well as in Xenopus oocytes. Because insulin and IGF-1 effects on meiosis are inhibited by wortmannin (29Liu X.J. Sorisky A. Zhu L. Pawson T. Mol. Cell. Biol. 1995; 15: 3563-3570Crossref PubMed Scopus (70) Google Scholar), PI3-K has been implicated in oocyte maturation. Injection of a constitutively active PI3-K mRNA causes resumption of meiosis (30Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (516) Google Scholar), whereas injection of the lipid phosphatase SIP/SHIP blocks the insulin effects (31Deuter-Reinhard M. Apell G. Pot D. Klippel A. Williams L.T. Kavanaugh W.M. Mol. Cell. Biol. 1997; 17: 2559-2565Crossref PubMed Scopus (60) Google Scholar). Our data extend these observations by identifying downstream steps in the pathway by which IGF-1 induces resumption of meiosis. As for somatic cells where the PI3-K effects are mediated by activation of Akt, we have shown that Akt activates resumption of meiosis in a manner similar to insulin/IGF-1.Although the myr-Akt efficiently promoted oocyte maturation, a wild type Akt was only partially effective in the oocyte model, consistent with observations in mammalian cells showing that wild type Akt has low basal activity in the absence of insulin or growth factor signals (7Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar,8Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 12Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 13Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar). Unlike the myr-Akt, the A2myr-Akt lacking the PH domain and with a mutation in the myristoylation signal was ineffective in inducing resumption of meiosis. This protein was efficiently expressed, and the overall Akt activity recovered in oocyte extracts was comparable with that obtained by expression of myr-Akt. When the activity of A2myr-Akt was corrected for the amount of protein expressed, this mutant kinase had one-fourth the specific activity of the myr-Akt. Because the only difference between A2myr-Akt and myr-Akt is the ability to interact with the lipid bilayer, we can conclude that Akt interaction with lipids is essential for the signaling meiotic resumption.Our findings that PDE3 inhibitors block the oocyte maturation induced by insulin and Akt, but not progesterone, indicate that a PDE3 is involved in the PI3-K signal transduction pathway activated by IGF-1/insulin. In agreement with this hypothesis, we have shown that insulin, PI3-K, and Akt stimulate the activity of PDE3 endogenous to the oocyte. That Akt activation of PDE3 may be a sufficient signal for meiosis resumption is strongly suggested by the observation that expression of a PDE causes GVBD. Our conclusion is consistent with the finding in adipocytes, where it has been shown that insulin through the PI3-K pathway activates PDE3B (32Degerman E. Belfrage P. Manganiello V.C. Biochem. Soc. Trans. 1996; 24: 1010-1014Crossref PubMed Scopus (27) Google Scholar).Our data do not exclude the possibility that Akt is at a branch point in the signaling pathway controlling maturation. Upon activation, this kinase may phosphorylate several substrates in addition to a PDE3, and the activation of a PDE3 has only a permissive role on meiotic maturation. According to this model, both the release of a cAMP-dependent blockade and the activation of a distinct signaling cascade may be required for meiotic resumption. For instance, Akt activates the p70 ribosomal S6 kinase, which may be involved in the regulation of mos translation, a crucial step in oocyte meiosis (33Sagata N. Oskarsson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-525Crossref PubMed Scopus (462) Google Scholar).Regardless of the mechanism of Akt action, our findings indicate a role for this enzyme in oocyte maturation. Interestingly, the cloning of 3-phosphoinositide-dependent kinase-1, the kinase-phosphorylating Thr-308 of Aktα, has uncovered a structural and function homology with the Drosophila DSTPK61 kinase. Although little is known about the function of the PI3-K and Akt pathway in Drosophila, DSTPK61 kinase may play an important role in the differentiation of the female and male germ cells (9Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). Our data in the Xenopus oocyte are in line with the view that this pathway is involved in oocyte maturation. The physiological signals activating the PI3-K and the Akt pathway are unknown at present but may be a means by which somatic cells control the function of germ cells. It is worth noting that the PDE3A mRNA (34Tsafriri A. Chun S.Y. Zhang R. Hsueh A.J. Conti M. Dev. Biol. 1996; 178: 393-402Crossref PubMed Scopus (266) Google Scholar) and protein are expressed in mammalian oocytes. Consistent with that shown inXenopus, PDE3 inhibitors block the spontaneous resumption of meiosis in rat and mouse oocytes as well as the maturation induced by the luteinizing hormone in the intact follicle (34Tsafriri A. Chun S.Y. Zhang R. Hsueh A.J. Conti M. Dev. Biol. 1996; 178: 393-402Crossref PubMed Scopus (266) Google Scholar, 35Wiersma A. Hirsh B. Tsafriri A. Hanssen R.J.G.M. Van de Kant M. Kloosterboer H.J. Conti M. Hseuh A.J.W. XIth International Workshop on Development and Function of the Reproductive Organs. 1998; (Amsterdam, Netherlands): 15Google Scholar). Recentin vivo observations have further confirmed the crucial role of PDE3 for resumption of meiosis in mammals (35Wiersma A. Hirsh B. Tsafriri A. Hanssen R.J.G.M. Van de Kant M. Kloosterboer H.J. Conti M. Hseuh A.J.W. XIth International Workshop on Development and Function of the Reproductive Organs. 1998; (Amsterdam, Netherlands): 15Google Scholar). Thus, our data on the Xenopus oocyte open the possibility that, in the mammalian follicle, physiological signals promoting resumption of meiosis regulate the intraoocyte cAMP levels via the PI3-K/Akt signaling pathway. Akt is a serine-threonine kinase in the PI3-K signaling pathway that mediates growth factor regulation of cell differentiation and survival (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar). Here we provide evidence for an additional role of this Akt kinase in the control of the cell cycle and meiosis. Akt expression in the Xenopus oocyte causes germinal vesicle breakdown and mitogen-activated protein kinase phosphorylation, hallmarks of the resumption of meiosis. Furthermore, our findings indicate that a PDE3 is distal to Akt activation. This conclusion is supported by both the pharmacological manipulation of the oocyte PDE and by the observation that an increase in PDE activity follows expression of the Akt kinase in oocyte. Finally, the finding that expression of a PDE in the oocyte per se is sufficient to promote a meiotic resumption is consistent with the above conclusions. Numerous observations have underscored the importance of the lipid PI3-K in insulin and growth factor signaling in somatic cells (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar) as well as in Xenopus oocytes. Because insulin and IGF-1 effects on meiosis are inhibited by wortmannin (29Liu X.J. Sorisky A. Zhu L. Pawson T. Mol. Cell. Biol. 1995; 15: 3563-3570Crossref PubMed Scopus (70) Google Scholar), PI3-K has been implicated in oocyte maturation. Injection of a constitutively active PI3-K mRNA causes resumption of meiosis (30Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (516) Google Scholar), whereas injection of the lipid phosphatase SIP/SHIP blocks the insulin effects (31Deuter-Reinhard M. Apell G. Pot D. Klippel A. Williams L.T. Kavanaugh W.M. Mol. Cell. Biol. 1997; 17: 2559-2565Crossref PubMed Scopus (60) Google Scholar). Our data extend these observations by identifying downstream steps in the pathway by which IGF-1 induces resumption of meiosis. As for somatic cells where the PI3-K effects are mediated by activation of Akt, we have shown that Akt activates resumption of meiosis in a manner similar to insulin/IGF-1. Although the myr-Akt efficiently promoted oocyte maturation, a wild type Akt was only partially effective in the oocyte model, consistent with observations in mammalian cells showing that wild type Akt has low basal activity in the absence of insulin or growth factor signals (7Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar,8Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 12Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 13Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar). Unlike the myr-Akt, the A2myr-Akt lacking the PH domain and with a mutation in the myristoylation signal was ineffective in inducing resumption of meiosis. This protein was efficiently expressed, and the overall Akt activity recovered in oocyte extracts was comparable with that obtained by expression of myr-Akt. When the activity of A2myr-Akt was corrected for the amount of protein expressed, this mutant kinase had one-fourth the specific activity of the myr-Akt. Because the only difference between A2myr-Akt and myr-Akt is the ability to interact with the lipid bilayer, we can conclude that Akt interaction with lipids is essential for the signaling meiotic resumption. Our findings that PDE3 inhibitors block the oocyte maturation induced by insulin and Akt, but not progesterone, indicate that a PDE3 is involved in the PI3-K signal transduction pathway activated by IGF-1/insulin. In agreement with this hypothesis, we have shown that insulin, PI3-K, and Akt stimulate the activity of PDE3 endogenous to the oocyte. That Akt activation of PDE3 may be a sufficient signal for meiosis resumption is strongly suggested by the observation that expression of a PDE causes GVBD. Our conclusion is consistent with the finding in adipocytes, where it has been shown that insulin through the PI3-K pathway activates PDE3B (32Degerman E. Belfrage P. Manganiello V.C. Biochem. Soc. Trans. 1996; 24: 1010-1014Crossref PubMed Scopus (27) Google Scholar). Our data do not exclude the possibility that Akt is at a branch point in the signaling pathway controlling maturation. Upon activation, this kinase may phosphorylate several substrates in addition to a PDE3, and the activation of a PDE3 has only a permissive role on meiotic maturation. According to this model, both the release of a cAMP-dependent blockade and the activation of a distinct signaling cascade may be required for meiotic resumption. For instance, Akt activates the p70 ribosomal S6 kinase, which may be involved in the regulation of mos translation, a crucial step in oocyte meiosis (33Sagata N. Oskarsson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-525Crossref PubMed Scopus (462) Google Scholar). Regardless of the mechanism of Akt action, our findings indicate a role for this enzyme in oocyte maturation. Interestingly, the cloning of 3-phosphoinositide-dependent kinase-1, the kinase-phosphorylating Thr-308 of Aktα, has uncovered a structural and function homology with the Drosophila DSTPK61 kinase. Although little is known about the function of the PI3-K and Akt pathway in Drosophila, DSTPK61 kinase may play an important role in the differentiation of the female and male germ cells (9Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). Our data in the Xenopus oocyte are in line with the view that this pathway is involved in oocyte maturation. The physiological signals activating the PI3-K and the Akt pathway are unknown at present but may be a means by which somatic cells control the function of germ cells. It is worth noting that the PDE3A mRNA (34Tsafriri A. Chun S.Y. Zhang R. Hsueh A.J. Conti M. Dev. Biol. 1996; 178: 393-402Crossref PubMed Scopus (266) Google Scholar) and protein are expressed in mammalian oocytes. Consistent with that shown inXenopus, PDE3 inhibitors block the spontaneous resumption of meiosis in rat and mouse oocytes as well as the maturation induced by the luteinizing hormone in the intact follicle (34Tsafriri A. Chun S.Y. Zhang R. Hsueh A.J. Conti M. Dev. Biol. 1996; 178: 393-402Crossref PubMed Scopus (266) Google Scholar, 35Wiersma A. Hirsh B. Tsafriri A. Hanssen R.J.G.M. Van de Kant M. Kloosterboer H.J. Conti M. Hseuh A.J.W. XIth International Workshop on Development and Function of the Reproductive Organs. 1998; (Amsterdam, Netherlands): 15Google Scholar). Recentin vivo observations have further confirmed the crucial role of PDE3 for resumption of meiosis in mammals (35Wiersma A. Hirsh B. Tsafriri A. Hanssen R.J.G.M. Van de Kant M. Kloosterboer H.J. Conti M. Hseuh A.J.W. XIth International Workshop on Development and Function of the Reproductive Organs. 1998; (Amsterdam, Netherlands): 15Google Scholar). Thus, our data on the Xenopus oocyte open the possibility that, in the mammalian follicle, physiological signals promoting resumption of meiosis regulate the intraoocyte cAMP levels via the PI3-K/Akt signaling pathway. We thank Dr. Jim Ferrell for the polyclonal anti-ERK2 antibody and the helpful discussion; Eric Machleder and Mike Wu for help with microinjections; Morris Birnbaum for iSH2-p110 clone; and Kristina Kovacina and Aimee Kohn for pECE-Akt constructs.
In this study, we synergistically integrate Ro5's target evaluation (SpectraView) and deep-learning-driven virtual screening (HydraScreen) tools with Strateos automated robotic cloud lab optimized for ultra high-throughput screening, to experimentally validate Ro5's tools. This integrated approach leads to a significant acceleration in the processes of target identification and hit discovery. By using SpectraView to select IRAK1 as the focal point of our investigation, we prospectively validate HydraScreen structure-based deep learning model. We can achieve the identification of an 23.8% of all IRAK1 hits within the top 1% of ranked compounds. HydraScreen also outperforms traditional virtual screening techniques and offers advanced features such as ligand pose confidence scoring. Simultaneously, we identify three potent (nanomolar) scaffolds from our compound library, two of which represent novel candidates for IRAK1 and hold potential for future development. Our platforms and innovative tools promise to expedite the early stages of drug discovery.
Background: Target identification and hit identification can be transformed through the application of biomedical knowledge analysis, AI-driven virtual screening and robotic cloud lab systems. However there are few prospective studies that evaluate the efficacy of such integrated approaches. Results: We synergistically integrate our in-house-developed target evaluation (SpectraView) and deep-learning-driven virtual screening (HydraScreen) tools with an automated robotic cloud lab designed explicitly for ultra-high-throughput screening, enabling us to validate these platforms experimentally. By employing our target evaluation tool to select IRAK1 as the focal point of our investigation, we prospectively validate our structure-based deep learning model. We can identify 23.8% of all IRAK1 hits within the top 1% of ranked compounds. The model outperforms traditional virtual screening techniques and offers advanced features such as ligand pose confidence scoring. Simultaneously, we identify three potent (nanomolar) scaffolds from our compound library, 2 of which represent novel candidates for IRAK1 and hold promise for future development. Conclusion: This study provides compelling evidence for SpectraView and HydraScreen to provide a significant acceleration in the processes of target identification and hit discovery. By leveraging Ro5's HydraScreen and Strateos' automated labs in hit identification for IRAK1, we show how AI-driven virtual screening with HydraScreen could offer high hit discovery rates and reduce experimental costs. Scientific contribution: We present an innovative platform that leverages Knowledge graph-based biomedical data analytics and AI-driven virtual screening integrated with robotic cloud labs. Through an unbiased, prospective evaluation we show the reliability and robustness of HydraScreen in virtual and high-throughput screening for hit identification in IRAK1. Our platforms and innovative tools can expedite the early stages of drug discovery.
cyclodextrin glycosyltransferases and α‐amylases are two groups of enzymes with related secondary structures. However, cyclodextrin glycosyltransferases display transferase activities not present in α‐amylases, probably derived from the existence of two more domains and different amino acid sequences. The hydrolytic activity of cyclodextrin glycosyltransferases is generally quite low, except for two cyclodextrin glycosyltransferases from termophiles. In this work, we have carried out the chemical modification (with acetic anhydride) of the amino groups of cyclodextrin glycosyltransferase from Thermoanaerobacter to assess their contributions to protein function. The acetylated cyclodextrin glycosyltransferase showed a significant reduction of its cyclization, coupling and disproportionation activities. Surprisingly, the hydrolytic (saccharifying) activity was slightly enhanced. These results suggest the participation of one or more lysine side chains in the interactions contributing to the transferase activity, either in any of the S n subsites or in the acceptor binding site.
Abstract The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.
A series of potent and selective [1,2,4]triazolo[1,5-a]pyrimidine PDE2a inhibitors is reported. The design and improvement of the binding properties of this series was achieved using X-ray crystal structures in conjunction with careful analysis of electronic and structural requirements for the PDE2a enzyme. One of the lead compounds, compound 27 (DNS-8254), was identified as a potent and highly selective PDE2a enzyme inhibitor with favorable rat pharmacokinetic properties. Interestingly, the increased potency of compound 27 was facilitated by the formation of a halogen bond with the oxygen of Tyr827 present in the PDE2a active site. In vivo, compound 27 demonstrated significant memory enhancing effects in a rat model of novel object recognition. Taken together, these data suggest that compound 27 may be a useful tool to explore the pharmacology of selective PDE2a inhibition.
Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)8-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.
