β-Nicotinamide adenine dinucleotide (β-NAD) is a pivotal metabolite for all living organisms and functions as a diffusible electron acceptor and carrier in the catabolic arms of metabolism1,2. Furthermore, β-NAD is involved in diverse epigenetic, immunological and stress-associated processes, where it is known to be sacrificially utilized as an ADP-ribosyl donor for protein and DNA modifications, or the generation of cell-signalling molecules3,4. Here we report the function of β-NAD in secondary metabolite biosynthetic pathways, in which the nicotinamide dinucleotide framework is heavily decorated and serves as a building block for the assembly of a novel class of natural products. The gatekeeping enzyme of the discovered pathway (SbzP) catalyses a pyridoxal phosphate-dependent [3+2]-annulation reaction between β-NAD and S-adenosylmethionine, generating a 6-azatetrahydroindane scaffold. Members of this novel family of β-NAD-tailoring enzymes are widely distributed in the bacterial kingdom and are encoded in diverse biosynthetic gene clusters. The findings of this work set the stage for the discovery and exploitation of β-NAD-derived natural products.
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Data that support the findings of this study are available within the paper and its Supplementary Information, or are available from the corresponding authors upon request.
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This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI grant numbers JP16H06443, JP17H04763, JP19H04641, JP20H00490, JP20KK0173 and JP21K18246), the New Energy and Industrial Technology Development Organization (NEDO, Grant Number JPNP20011) and AMED (Grant Number JP21ak0101164), Japan Science and Technology Agency (JST SICORP grant number JPMJSC1701), UTEC-UTokyo FSI Research Grant Program, Mochida Memorial Foundation for Medical and Pharmaceutical Research, and Takeda Science Foundation, Kato Memorial Bioscience Foundation, The Asahi Glass Foundation, and a Sir Charles Hercus Fellowship (Health Research Council of New Zealand) to G.B. (17/058). We thank GlaxoSmithKline for providing Streptomyces sp. NCIMB 40513; G. Cao for providing S. gilvosporeus F607; M. Kobayashi for providing pHSA81; Deutsche Forschungsgemeinschaft (DFG BA 6870/1-1) for a postdoctoral research fellowship to L.B.; T. Mori and R. Ushimaru for critical reading of the manuscript; F. Kudo, T. Eguchi and T. Ueda for instruction on the stopped flow apparatus; and H. Zhang, F. Hayashi and Y. Ishii for measurement of NMR spectra at RIKEN Yokohama.
The authors declare no competing interests.
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Extended data figures and tables
a, LC-MS analysis of single gene expression culture extracts (UV trace 340 nm). b, Summary of isotope feeding experiment results and retrobiosynthetic considerations. c, LC-MS analysis of the productive SbzP substrate combination of β-NAD (5) and SAM (6) with concomitant appearance of dinucleotide product 7 (UV trace 258 nm). “Control” indicates incubation of substrates without enzyme.
a, LC-MS analysis of gene deletion strain culture extracts (UV trace 280 nm). b, LC-MS analysis of SbzQ-mediated conversion of 7 to 8 (EIC traces of substrate and product). c, LC-MS analysis of SbzI reaction of 8 to 9 (UV trace 340 nm). d, LC-MS analysis of deadenosylribosylation catalyzed by SbzN+O+H (EIC traces of substrate and product). e, LC-MS analysis of F420-dependent reduction of 11 to 12 (EIC traces of substrate and products). f, LC-MS analysis of SbzE-mediated methylation towards 1 (UV trace 287 nm). g, Proposed steps in the deadenosylribosylation reaction. “Control” indicates incubation of substrates without enzyme(s).
Extended Data Fig. 3 SbzP as a functionally and phylogenetically distinct PLP-dependent enzyme family.
a, Time-resolved photospectroscopic changes of SbzP after addition of SAM (6) (see Supplementary Note 5 for further details). b, Phylogenetic analysis of SbzP and reported subfamily members of the AAT-I protein superfamily.
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Barra, L., Awakawa, T., Shirai, K. et al. β-NAD as a building block in natural product biosynthesis. Nature 600, 754–758 (2021). https://doi.org/10.1038/s41586-021-04214-7