Publications
Highlighted Publications
(Lane Lab students denoted with *)
Deletti G*, Green SD*, Weber C*, Patterson KN*, Joshi SS*, Khopade TM, Coban M, Veek-Wilson J*, Caulfield T, Viswanathan R, Lane AL. (2023) Unveiling an indole alkaloid diketopiperazine biosynthetic pathway that features a unique stereoisomerase and multifunctional methyltransferase.
Nature Communications. 14: 2558
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Abstract: The 2,5-diketopiperazines are a prominent class of bioactive molecules. The nocardioazines are actinomycete natural products that feature a pyrroloindoline diketopiperazine scaffold composed of two D-tryptophan residues functionalized by N- and C-methylation, prenylation, and diannulation. Here we identify and characterize the nocardioazine B biosynthetic pathway from marineNocardiopsis sp. CMB-M0232 by using heterologous biotransformations, in vitro biochemical assays, and macromolecular modeling. Assembly of the cyclo-L-Trp-L-Trp diketopiperazine precursor is catalyzed by a cyclodipeptide synthase. A separate genomic locus encodes tailoring of this precursor and includes an aspartate/glutamate racemase homolog as an unusual D/Lisomerase acting upon diketopiperazine substrates, a phytoene synthase-like prenyltransferase as the catalyst of indole alkaloid diketopiperazine prenylation, and a rare dual function methyltransferase as the catalyst of both N- and C-methylation as the final steps of nocardioazine B biosynthesis. The biosynthetic paradigms revealed herein showcase Nature’s molecular ingenuity and lay the foundation for diketopiperazine diversification via biocatalytic approaches.
Borgman P*, Lopez R*, Lane AL. (2019) The expanding spectrum of diketopiperazine natural product pathways containing cyclodipeptide synthases.
Organic & Biomolecular Chemistry. 17: 2305-2314
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Abstract: Microorganisms are remarkable chemists, with enzymes as their tools for executing multi-step syntheses to yield myriad natural products. Microbial synthetic aptitudes are illustrated by the structurally diverse 2,5-diketopiperazine (DKP) family of bioactive nonribosomal peptide natural products. Nonribosomal peptide synthetases (NRPSs) have long been recognized as catalysts for formation of DKP scaffolds from two amino acid substrates. Cyclodipeptide synthases (CDPSs) are more recently recognized catalysts of DKP assembly, employing two aminoacyl-tRNAs (aa-tRNAs) as substrates. CDPS-encoding genes are typically found in genomic neighbourhoods with genes encoding additional biosynthetic enzymes. These include oxidoreductases, cytochrome P450s, prenyltransferases, methyltransferases, and cyclases, which equip the DKP scaffold with groups that diversify chemical structures and confer biological activity. These tailoring enzymes have been characterized from nine CDPS-containing biosynthetic pathways to date, including four during the last year. In this review, we highlight these nine DKP pathways, emphasizing recently characterized tailoring reactions and connecting new developments to earlier findings. Featured pathways encompass a broad spectrum of chemistry, including the formation of challenging C–C and C–O bonds, regioselective methylation, a unique indole alkaloid DKP prenylation strategy, and unprecedented peptide-nucleobase bond formation. These CDPS-containing pathways also provide intriguing models of metabolic pathway evolution across related and divergent microorganisms, and open doors to synthetic biology approaches for generation of DKP combinatorial libraries. Further, bioinformatics analyses support that much unique genetically encoded DKP tailoring potential remains unexplored, suggesting opportunities for further expansion of Nature's biosynthetic spectrum. Together, recent studies of DKP pathways demonstrate the chemical ingenuity of microorganisms, highlight the wealth of unique enzymology provided by bacterial biosynthetic pathways, and suggest an abundance of untapped biosynthetic potential for future exploration.
James ED*, Knuckley BA, Alqahtani N, Porwal S, Ban J*, Karty JA, Viswanathan R, Lane AL. (2016) Two different cyclodipeptide synthases from a marine actinomycete catalyze biosynthesis of the same diketopiperazine natural product.
ACS Synthetic Biology. 5: 547-553.
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Abstract: Diketopiperazine natural products are structurally diverse and offer many biological activities. Cyclodipeptide synthases (CDPSs) were recently unveiled as a novel enzyme family that employs aminoacyl-tRNAs as substrates for 2,5-diketopiperazine assembly. Here, the Nocardiopsis sp. CMB-M0232 genome is predicted to encode two CDPSs, NozA and NcdA. Metabolite profiles from E. coli expressing these genes and assays with purified recombinant enzymes revealed that NozA and NcdA catalyze cyclo(l-Trp-l-Trp) (1) biosynthesis from tryptophanyl-tRNA and do not accept other aromatic aminoacyl-tRNA substrates. Fidelity is uncommon among characterized CDPSs, making NozA and NcdA important CDPS family additions. Further, 1 was previously supported as a biosynthetic precursor of the nocardioazines; the current study suggests that Nocardiopsis sp. may derive this precursor from both NozA and NcdA. This study offers a rare example of a single bacterium encoding multiple phylogenetically distinct enzymes that yield the same secondary metabolite and provides tools for chemoenzymatic syntheses of indole alkaloid diketopiperazines.
Alqahtani N, Porwal SK, James ED*, Bis DM*, Karty JA, Lane AL, Viswanathan R. (2015) Synergism between Genome Sequencing, Tandem Mass Spectrometry, and Bio-Inspired Synthesis Reveals Insights into Nocardioazine B Biogenesis.
Organic & Biomolecular Chemistry. 13: 7177-7192. (Featured on cover)
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Abstract: Marine actinomycete-derived natural products continue to inspire chemical and biological investigations. Nocardioazines A and B (3 and 4), from Nocardiopsis sp. CMB-M0232, are structurally unique alkaloids featuring a 2,5-diketopiperazine (DKP) core functionalized with indole C3-prenyl as well as indole C3- and N-methyl groups. The logic of their assembly remains cryptic. Bioinformatics analyses of the Nocardiopsis sp. CMB-M0232 draft genome afforded the noz cluster, split across two regions of the genome, and encoding putative open reading frames with roles in nocardioazine biosynthesis, including cyclodipeptide synthase (CDPS), prenyltransferase, methyltransferase, and cytochrome P450 homologs. Heterologous expression of a twelve gene contig from the noz cluster in Streptomyces coelicolor resulted in accumulation of cyclo-L-Trp-L-Trp DKP (5). This experimentally connected the noz cluster to indole alkaloid natural product biosynthesis. Results from bioinformatics analyses of the noz pathway along with challenges in actinomycete genetics prompted us to use asymmetric synthesis and mass spectrometry to determine biosynthetic intermediates in the noz pathway. The structures of hypothesized biosynthetic intermediates 5 and 12–17 were firmly established through chemical synthesis. LC-MS and MS-MS comparison of these synthetic compounds with metabolites present in chemical extracts from Nocardiopsis sp. CMB-M0232 revealed which of these hypothesized intermediates were relevant in the nocardioazine biosynthetic pathway. This established the early and mid-stages of the biosynthetic pathway, demonstrating that Nocardiopsisperforms indole C3-methylation prior to indole C3-normal prenylation and indole N1′-methylation in nocardioazine B assembly. These results highlight the utility of merging bioinformatics analyses, asymmetric synthetic approaches, and mass spectrometric metabolite profiling in probing natural product biosynthesis.
Bis DM*, Ban YH*, James ED*, Alqahtani N, Viswanathan R, Lane AL. (2015) Characterization of the Nocardiopsin Biosynthetic Gene Cluster Reveals Similarities to and Differences from the Rapamycin and FK-506 Pathways.
ChemBioChem. 16: 990-997.
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Abstract: Macrolide-pipecolate natural products, such as rapamycin (1) and FK-506 (2), are renowned modulators of FK506-binding proteins (FKBPs). The nocardiopsins, from Nocardiopsis sp. CMB-M0232, are the newest members of this structural class. Here, the biosynthetic pathway for nocardiopsins A–D (4–7) is revealed by cloning, sequencing, and bioinformatic analyses of the nsn gene cluster. In vitro evaluation of recombinant NsnL revealed that this lysine cyclodeaminase catalyzes the conversion of L-lysine into theL-pipecolic acid incorporated into 4 and 5. Bioinformatic analyses supported the conjecture that a linear nocardiopsin precursor is equipped with the hydroxy group required for macrolide closure in a previously unobserved manner by employing a P450 epoxidase (NsnF) and limonene epoxide hydrolase homologue (NsnG). The nsn cluster also encodes candidates for tetrahydrofuran group biosynthesis. The nocardiopsin pathway provides opportunities for engineering of FKBP-binding metabolites and for probing new enzymology in nature's polyketide tailoring arsenal.
● Khopade TM, Ajayan K, Vincent DM, Lane AL, Viswanathan R. (2022) Biomimetic Total Synthesis of (+)-Nocardioazine B and Analogs. Journal of Organic Chemistry. 87: 11519-11533
● Lane AL. (2021) Review of Molecular Evolutionary Models in Drug Discovery. Journal of Natural Products. 84: 1420.
● Khopade T, Ajayan K, Joshi SS*, Lane AL, Viswanathan R. (2021) Bioinspired Bronsted-Acid-Promoted Regioselective Tryptophan Isoprenylations. ACS Omega. 6: 10840-10858.
● Caulfield TR, Hayes KE, Qiu Y, Coban M, Oh JS*, Lane AL, Yoshimitsu T, Hazlehurst L, Copland JA, Tun HW. (2020) A virtual screening platform identifies chloroethylagelastatin A as a potential ribosomal inhibitor. Biomolecules. 10: 1407.
● Protasov ES*, Axenov-Gribanov DV, Shatilina ZM, Timofeyev MV, Lane AL. (2020) Freshwater Actinobacteria from Sediments of the Deep and Ancient Lake Baikal and their Genetic Potential as Producers of Secondary Metabolites. Aquatic Microbial Ecology. 84: 1-14.
● Carey J*, Nguyen T*, Korchak J*, Beecher C, de Jong F, Lane AL. (2019) An Isotopic Ratio Outlier Analysis Approach for Global Metabolomics of Biosynthetically Talented Actinomycetes. Metabolites. 9: 181.
● Lundy TA*, Lane AL. (2018) Interactions between microorganisms as modulators of natural product biosynthesis. In: Chemical Ecology: The Ecological Impacts of Marine Natural Products. Puglisi MP and Becerro M Eds. CRC Press.
● Von Roemeling CA, Caulfield TR, Marlow L, Bok I, Wen J, Miller JL, Hughes R*, Hazlehurst L, Pinkerton AB, Radisky DC, Tun H, Kim YSB, Lane AL, Copland JA. (2018) Accelerated bottom-up drug design platform enables the design of novel stearoyl CoA desaturase 1 inhibitors for cancer therapy. Oncotarget. 9: 3-20.
● Trevathan-Tackett SM, Lane AL, Bishop N, Ross CP. (2015) Metabolites derived from the tropical sea grass Thalassia testudinum are bioactive against pathogenic Labyrinthula sp. Aquatic Botany. 122: 1-8.
● Peterson M*, Lane AL, Ahearn GA. (2015) Analysis of glycylsarcosine transport by lobster intestine using gas chromatography. Journal of Comparative Physiology – B. 185: 37-45.
● Lane AL, Mandelare PE*, Ban YH*. (2015) New developments in NMR methodologies with special roles in natural product discovery. In: Applications of NMR Spectroscopy, Volume 3. Rahman AU and Choudhary I, Eds. Bentham eBook. pp. 79-117.
● Abdul-Hay SO, Lane AL, Caulfield TR, Claussin C, Bertrand J, Masson A, Choudhry S, Fauq AH, Maharvi GM, Leissring MA. (2013) Optimization of peptide hydroxamate inhibitors of insulin-degrading enzyme reveals marked substrate selectivity. Journal of Medicinal Chemistry. 56: 2246-2255.
● Lane AL, Moore BS. (2011) A sea of biosynthesis: Marine natural products meet the molecular age. Natural Product Reports. 28: 411-428.