18 マクロラクタムポリケチド抗生物質ビセニスタチンの生合成におけるアミノ酸スターター部位の構築機構(口頭発表の部)

抄録

Vicenistatin is a 20-membered macrolactam antibiotic isolated from Streptomyces halstedii HC34. Its hybrid structure consisting of a unique amino acid (3-amino-2-methylpropionate, AmP) and polyketide is characteristic and thus prompted us to investigate the biosynthetic pathway, especially concerning the construction of the unique amino acid and its combination with the polyketide pathway. Our previous incorporation study indicated that the AmP moiety of vicenistatin is derived from L-glutamate (Glu) via 3-methylaspartate (MeAsp). Also, since the free form of AmP was not incorporated into the moiety, MeAsp seemed to be modified as CoA or ACP thioester before decarboxylation. Further, bioinfomatic analysis of the vicenistatin biosynthetic (yin) gene cluster revealed that eight unique proteins (VinH, I, J, K, L, M, N and 0) seemed to be responsible for the unique amino acid biosynthesis accompanying four PKSs and seven deoxysugar biosynthetic enzymes. In this report, we characterized the eight unique proteins, which were all involved in the AmP biosynthesis and the connection of the generated AmP with the polyketide pathway expectedly. At first, inactivation of the vinl gene for glutamate mutase E subunit resulted in the loss of vicenistain production, while the disruptant recovered it when MeAsp was added into the culture. This result suggested that glutamate mutase S subuint VinH and E subuint VinI generate MeAsp from Glu cooperatively. To investigate the other six proteins (VinJ, K, L, M, N and 0), we prepared the recombinant enzymes, which were successfully expressed in Escherichia coli. Two ATP-dependent ligases VinM and VinN were predicted to act as freestanding adenylation domains that catalyze two half reactions: The first is ATP-dependent activation of carboxyl group of amino acid and the second half-reaction is the transfer of the aminoacyl moiety of the adenylate intermediate to ACP (VinL) or its equivalent. Based on typical photometric assay by detection of released pyrophosphate, VinM was found to recognize small size of amino acids such as alanine, glycine and serine. On the other hands, VinN specifically recognized MeAsp. Futher, when the holo form of VinL was added into the VinN reaction mixture with ATP and MeAsp, the expected MeAsp-VinL formation was clearly detected by LC-ESI-MS analysis. Subsequently, the formed MeAsp-VinL was decarboxylated to be AmP-VinL by a PLP-dependent decarboxylase VinO. The generated AmP-VinL was then reacted with VinM, ATP and alanine to examine the additional attachment of aminoacyl group onto AmP-VinL to prevent a presumable thermodynamically favorable six-membered lactam formation during the chain elongation by PKS. As a result, a dipeptidyl ACP, Ala-AmP-VinL, was efficiently formed by VinM. The Ala-AmP-VinL was then used as a substrate for an acyltransferase VinK, which was speculated to catalyze the acyl transfer reaction between VinL and VinPl-ACP domain at the loading module of PKS. As we expected, the dipeptide transfer reaction occurred efficiently to give Ala-AmP-VinP 1 -ACP, which should be a starter molecule of vicenilactam PKS. Finally, to investigate the function of VinJ, a peptidase, we chemically synthesized the ethyl ester of N-alanyl-secovicenilactam as a substrate analog for the elongated polyketide intermediate. As we expected, VinJ catalyzed the hydrolysis of the analog to afford secovicenilactam ethyl ester, which was then macrocyclized by VinP4-TE to give vicnilactam to complete the biosynthesis. In this study, we clarified the unique starter biosynthesis of vicenistatin. It should be noteworthy that these amino acid carrying enzymes are highly conserved in the other macrolactam biosynthetic gene cluster, indicating that the clarified dipeptide strategy to carry amino acids appears to be a common way in the macrolactam biosynthesis. Thus, our finding shed a light on the rational design of engineered

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