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β-NAD as a building block in natural product biosynthesis


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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Fig. 1: The discovered β-NAD-utilizing sbz biosynthetic pathway.
Fig. 2: Proposed reaction mechanism of the SbzP-mediated [3+2]-annulation reaction.
Fig. 3: β-NAD-utilizing SbzP homologues.

Data availability

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.

Author information

Authors and Affiliations



T.A. and I.A. conceived the idea for the study. L.B., T.A. and I.A. developed the hypothesis and designed the experiments. L.B. and T.A. performed the in vivo and in vitro experiments. L.B. and K.S. performed compound isolation and characterization. L.B. and T.A. performed bioinformatic analysis to identify biosynthetic gene clusters and protein functions. L.B., T.A. and K.S. performed protein purification. G.B. expressed SbzF and provided the F420 cofactor regeneration system. T.A. and Z.H. performed the feeding experiments. All authors analysed and discussed the results. L.B., T.A. and I.A. prepared the manuscript.

Corresponding authors

Correspondence to Takayoshi Awakawa or Ikuro Abe.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Identification of the gatekeeping enzyme SbzP.

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.

Extended Data Fig. 2 Gene deletion-guided in vitro reconstitution of the sbz pathway.

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.

Supplementary information

Supplementary Information

This file contains Supplementary Text; Supplementary Figs. 1 – 81; Supplementary Table 1; Supplementary Notes 1–8 and Supplementary References

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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).

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