TY - JOUR

T1 - Ketoreductase domain dysfunction expands chemodiversity: malyngamide biosynthesis in the cyanobacterium Okeania hirsuta

AU - Moss, Nathan

AU - Leao, Tiago

AU - Rankin, Michael

AU - McCullough, Tyler M.

AU - Qu, Pingping

AU - Коробейников, Антон Иванович

AU - Smith, Janet L.

AU - Gerwick, Lena

AU - Gerwick, William H.

N1 - Funding Information: *E-mail: wgerwick@ucsd.edu. ORCID Janet L. Smith: 0000-0002-0664-9228 Lena Gerwick: 0000-0001-6108-9000 William H. Gerwick: 0000-0003-1403-4458 Funding This work was supported by Grants GM107550-03, NIH GM118815-01A1, NIH CA108874, NIH GM067550, NIH R01 DK042303, and the Margaret J. Hunter Collegiate Professorship. A.K. was supported by St. Petersburg State University Grant 15.61.951.2015. Notes The authors declare the following competing financial interest(s): W.G. and L.G. have an equity interest in Sirenas Marine Discovery. W.G. serves on the company's Scientific Advisory Board. The terms of this arrangement were reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies.

PY - 2018/12

Y1 - 2018/12

N2 - Dozens of type A malyngamides, principally identified by a decorated six-membered cyclohexanone head group and methoxylated lyngbic acid tail, have been isolated over several decades. Their environmental sources include macro- and microbiotic organisms, including sea hares, red alga, and cyanobacterial assemblages but their true producing organism has remained enigmatic. Many type A analogs display potent bioactivity in human-health related assays, spurring an interest in this molecular class and its biosynthetic pathway. Here we present the discovery of the type A malyngamide biosynthetic pathway in the first sequenced genome of the cyanobacterial genus Okeania. Bioinformatic analysis of two cultured Okeania genome assemblies identified 62 and 68 kb polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) pathways with unusual loading and termination genes. NMR data of malyngamide C acetate derived from 13C-substrate-fed cultures provided evidence that an intact octanoate moiety is transferred to the first KS module via a LipM homolog originally associated with lipoic acid metabolism and implicated an inactive ketoreductase (KR0) as critical for six-membered ring formation, a hallmark of the malyngamide family. Phylogenetic analysis and homology modeling of the penultimate KR0 domain inferred structural cofactor-binding and active site alterations as contributory to domain dysfunction, which was confirmed by recombinant protein expression and NADPH binding assay. The carbonyl retained from this KR0 ultimately enables an intramolecular Knoevenagel condensation to form the characteristic cyclohexanone ring. Understanding this critical step allows assignment of a biosynthetic model for all type A malyngamides, whereby well-characterized tailoring modifications explain the surprising proliferation and diversity of analogs.

AB - Dozens of type A malyngamides, principally identified by a decorated six-membered cyclohexanone head group and methoxylated lyngbic acid tail, have been isolated over several decades. Their environmental sources include macro- and microbiotic organisms, including sea hares, red alga, and cyanobacterial assemblages but their true producing organism has remained enigmatic. Many type A analogs display potent bioactivity in human-health related assays, spurring an interest in this molecular class and its biosynthetic pathway. Here we present the discovery of the type A malyngamide biosynthetic pathway in the first sequenced genome of the cyanobacterial genus Okeania. Bioinformatic analysis of two cultured Okeania genome assemblies identified 62 and 68 kb polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) pathways with unusual loading and termination genes. NMR data of malyngamide C acetate derived from 13C-substrate-fed cultures provided evidence that an intact octanoate moiety is transferred to the first KS module via a LipM homolog originally associated with lipoic acid metabolism and implicated an inactive ketoreductase (KR0) as critical for six-membered ring formation, a hallmark of the malyngamide family. Phylogenetic analysis and homology modeling of the penultimate KR0 domain inferred structural cofactor-binding and active site alterations as contributory to domain dysfunction, which was confirmed by recombinant protein expression and NADPH binding assay. The carbonyl retained from this KR0 ultimately enables an intramolecular Knoevenagel condensation to form the characteristic cyclohexanone ring. Understanding this critical step allows assignment of a biosynthetic model for all type A malyngamides, whereby well-characterized tailoring modifications explain the surprising proliferation and diversity of analogs.

KW - COLLECTION

KW - DISCOVERY

KW - GENE-CLUSTER

KW - IDENTIFICATION

KW - INHIBITOR

KW - LIPOIC ACID

KW - LYNGBYA-MAJUSCULA

KW - MECHANISM

KW - NATURAL-PRODUCTS

KW - SKELETON

UR - http://www.scopus.com/inward/record.url?scp=85058079313&partnerID=8YFLogxK

UR - http://pubs.acs.org/doi/10.1021/acschembio.8b00910

UR - http://www.mendeley.com/research/ketoreductase-domain-dysfunction-expands-chemodiversity-malyngamide-biosynthesis-cyanobacterium-okea

U2 - 10.1021/acschembio.8b00910

DO - 10.1021/acschembio.8b00910

M3 - Article

VL - 13

SP - 3385

EP - 3395

JO - ACS Chemical Biology

JF - ACS Chemical Biology

SN - 1554-8929

IS - 12

ER -