Sequence, Cloning, and Analysis of the Fluvirucin B₁ Polyketide Synthase from Actinomadura vulgaris

Abstract

Fluvirucin B1, produced by Actinomadura vulgaris, is a 14-membered macrolactam active against a variety of infectious fungi as well as influenza A. Despite considerable interest from the synthetic community, very little information is available regarding the biosynthetic origins of the fluvirucins. Herein, we report the identification and initial characterization of the fluvirucin B1 polyketide synthase and related enzymes. The cluster consists of five extender modules flanked by an N-terminal acyl carrier protein and C-terminal thioesterase domain. All but one of the synthase modules contain the full complement of tailoring domains (ketoreductase, dehydratase, and enoyl reductase) as determined by sequence homology with known polyketide synthases. Acitve site analyses of several key components of the cluster are performed to further verify that this gene cluster is associated with production of fluvirucin B1. This work will both open doors toward a better understanding of macrolactam formation and provide an avenue to genetics-based diversification of fluvirucin structure.

Identification of the Fluvirucin B2 (Sch 38518) Biosynthetic Gene Cluster from Actinomadura fulva subsp. indica ATCC 53714: substrate Specificity of the β-Amino Acid Selective Adenylating Enzyme FlvN

“…The presence of three PKS genes, flvP1, flvP2, and flvP3, in the gene cluster is consistent with that of fluA, fluB, and fluC in the biosynthetic gene cluster of fluvirucin B 1 . 19) The module 1 of FlvP1, the modules 3 and 4 of FlvP2, and the module 5 of FlvP3 have a full set of PKS domains consisting β-ketosynthase (KS), acyltransferase (AT), dehydratase (DH), β-ketoreductase (KR), and enoylreductase (ER) domains, indicating that these PKS modules are responsible for constructing the fully reduced polyketide chain. The module 2 of FlvP1 would be responsible for constructing the C9-C10 moiety with a hydroxy group, because this module does not contain the DH domain.…”

“…Recently, the PKS genes responsible for the formation of fluvirucin B 1 aglycone were identified and characterized. 19) However, key β-amino acid-carrying enzymes including the VinN-type adenylation enzyme have not been identified in the reported fluvirucin B 1 biosynthetic gene cluster. The mechanism of the construction of the β-alanine moiety in fluvirucin thus remains elusive.…”

Biochemical Characterization of β‐Amino Acid Incorporation in Fluvirucin B2 Biosynthesis

“…Notable macrolactams include the fluvirucins (Flvs), rifamycin, and geldanamycin . Fluvirucin B 2 is a 14‐membered macrolactam that shows both antifungal and antiviral activity, and one of its main structural features is the β‐alanine starter unit of the polyketide skeleton (Scheme ). The biosynthetic nature of polyketide synthases and their potential to be reprogrammed could enhance existing chemically produced lactam analogues, such as valerolactam, caprolactam, and laurolactam (Scheme ).…”

Insights into the programmed ketoreduction of partially reducing polyketide synthases: stereo- and substrate-specificity of the ketoreductase domain

“…12 The synthetic method for 1d(S) and 1d(R) was adopted from a previously reported procedure and the structures of the products were established by MS and NMR spectroscopy. 60 For the synthesis of 2c, 3c and 4c, a 0.5 M solution of Meldrum's acid (1 eq.) in CH 2 Cl 2 was prepared in a round bottomed flask at room temperature.…”