Thioester reduction and aldehyde transamination are universal steps in actinobacterial polyketide alkaloid biosynthesis.

Open Access Article. Published on 22 August 2016. Downloaded on 17/03/2017 15:31:01. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Chemical Science View Article Online EDGE ARTICLE Cite this: Chem. Sci., 2017, 8, 411 View Journal | View Issue Thioester reduction and aldehyde transamination are universal steps in actinobacterial polyketide alkaloid biosynthesis† U. R. Awodi,‡ J. L. Ronan,‡ J. Masschelein,‡ E. L. C. de los Santos and G. L. Challis* Actinobacteria produce a variety of polyketide alkaloids with unusual structures. Recently, it was shown that a type I modular polyketide synthase (PKS) is involved in the assembly of coelimycin P1, a polyketide alkaloid produced by Streptomyces coelicolor M145. However, the mechanisms for converting the product of the PKS to coelimycin P1 remain to be elucidated. Here we show that the C-terminal thioester reductase (TR) domain of the PKS and an u-transaminase are responsible for release of the polyketide chain as an aldehyde and its subsequent reductive amination. Bioinformatics analyses identified numerous gene clusters in Received 24th June 2016 Accepted 21st August 2016 actinobacterial genomes that encode modular PKSs with a C-terminal TR domain and a homolog of the u-transaminase. These are predicted to direct the biosynthesis of both known and novel polyketide DOI: 10.1039/c6sc02803a alkaloids, suggesting that reductive chain release and transamination constitutes a conserved mechanism www.rsc.org/chemicalscience for the biosynthesis of such metabolites. Introduction An astonishing array of structurally-diverse polyketide metabolites with numerous applications in medicine, animal health and agriculture are produced by actinobacteria.1 Remarkable molecular machines known as type I modular polyketide synthases (PKSs) are responsible for the biosynthesis of the overwhelming majority of these natural products.2 Modular PKSs employ several distinct strategies for the creation of structural diversity, including the utilization of a variety of starter and extender units for assembly of the polyketide chain,3 diverse mechanisms for release of the fully-assembled chain from the PKS,4 and a wide range of on- and post-PKS tailoring reactions. Among these, the mechanism of chain release is arguably of prime importance in dening which structural class a metabolite belongs to. Thus, in macrolide biosynthesis chain release proceeds via macrolactonization,4 whereas in ansamycin biosynthesis the chain is released via macrolactamization;4 polyether biosynthesis employs hydrolytic chain release;4 (spiro)tetronates are assembled by condensation of the polyketide chain with a glyceryl thioester;5 and chain release in prodiginine biosynthesis proceeds via nucleophilic attack of an amino acid a-carbanion equivalent (see ESI† for further details).6 Incorporation experiments with isotope-labeled precursors have shown that several mono-, di-, tri- and tetracyclic alkaloids produced by actinobacteria, including latumcidin 1 (also known as abikoviromycin),7 nigrifactin 2,8 pyrindicin 3,9 cyclizidine 4,10 and streptazolin 5 (ref. 11) (Fig. 1) are derived from polyketide precursors. Several other actinobacterial alkaloids, such as streptazone E 6,12 4-hydroxy(2-penta-1,3-dienyl)piperidine 7,13 and indolizomycin 8 (ref. 14) can similarly be hypothesized to have a polyketide origin. In 2012, we reported the identication of the unusual alkaloid coelimycin P1 9 (Fig. 1) as the metabolic product of a cryptic polyketide (cpk) biosynthetic gene cluster in Streptomyces coelicolor M145.15 This Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK. E-mail: † Electronic supplementary information (ESI) available: Experimental procedures and additional tables, gures, chromatograms and results from biological assays. See DOI: 10.1039/c6sc02803a ‡ These authors contributed equally. This journal is © The Royal Society of Chemistry 2017 Fig. 1 Structures of polyketide alkaloids from actinobacteria. Chem. Sci., 2017, 8, 411–415 | 411 View Article Online Open Access Article. Published on 22 August 2016. Downloaded on 17/03/2017 15:31:01. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Chemical Science Fig. 2 Edge Article The proposed roles played by the CpkABC modular PKS and the CpkG u-transaminase in coelimycin P1 9 biosynthesis. provided the rst biochemical insights into the molecular mechanisms underlying actinobacterial polyketide alkaloid biosynthesis. The cpkA, cpkB and cpkC genes within the coelimycin biosynthetic gene cluster encode a hexamodular PKS, which is proposed on the basis of the predicted specicities of its catalytic domains to assemble thioester 10 (Fig. 2).15 This intermediate is thought to be released from the PKS by NAD(P)Hmediated reduction to the corresponding aldehyde 11, catalyzed by the thioester reductase (TR) domain at the C-terminus of CpkC.15 Reductive amination of aldehyde 11, catalyzed by the putative u-transaminase encoded by cpkG, is proposed to give amine 12 (or its (Z)-D-3,4 isomer, depending on the timing of D-2,3 isomerization).15 Elaboration of 12 to 9 is believed to proceed via a series of oxidations, a cyclodehydration and bisepoxide trapping by N-acetyl-L-cysteine, as illuminated by incorporation of labelled precursors.15 Results and discussion To experimentally investigate the role played by the CpkC TR domain in coelimycin 9 biosynthesis, it was overproduced in E. coli as an N-terminal His6 fusion protein and puried to homogeneity using nickel affinity chromatography. UHPLC-ESIQ-TOF-MS analysis conrmed that the puried protein had the expected molecular weight (see ESI†). The catalytic activity of the CpkC TR domain was investigated using octanoyl-CoA 13 as a mimic of the proposed natural substrate 10. NADH and NADPH consumption was examined by monitoring the decrease in absorbance at 340 nm. A substantial decrease in absorbance with time was observed when NADH was used, whereas no decrease in absorbance was observed for NADPH (Fig. 3), showing that octanoyl-CoA is reduced by the former but not the latter. No NADH consumption was observed in control reactions from which the enzyme was omitted (Fig. 3). In the absence of CpkG, which is proposed to intercept the octanal 14 formed by reduction of octanoyl-CoA 13 via transamination, CpkC-TR could catalyze further reduction of 14 to 1- 412 | Chem. Sci., 2017, 8, 411–415 Analysis of NAD(P)H consumption in the CpkC-TR-catalyzed reduction of octanoyl-CoA using a continuous spectrophotometric assay. Fig. 3 octanol 15 (Scheme 1). Thus, the reaction mixture containing CpkC-TR, octanoyl-CoA 13 and NADH was extracted with chloroform and derivatized with N,O-bis-(trimethylsilyl)acetamide (BSA) to convert any alcohols present to the corresponding trimethylsilyl (TMS) ether derivatives. GC-MS analysis of the derivatized (...truncated)