Functional assignment of multiple catabolic pathways for d-apiose

Abstract

Colocation of the genes encoding ABC, TRAP, and TCT transport systems and catabolic pathways for the transported ligand provides a strategy for discovering novel microbial enzymes and pathways. We screened solute-binding proteins (SBPs) for ABC transport systems and identified three that bind d-apiose, a branched pentose in the cell walls of higher plants. Guided by sequence similarity networks (SSNs) and genome neighborhood networks (GNNs), the identities of the SBPs enabled the discovery of four catabolic pathways for d-apiose with eleven previously unknown reactions. The new enzymes include d-apionate oxidoisomerase, which catalyzes hydroxymethyl group migration, as well as 3-oxo-isoapionate-4-phosphate decarboxylase and 3-oxo-isoapionate-4-phosphate transcarboxylase/hydrolase, which are RuBisCO-like proteins (RLPs). The web tools for generating SSNs and GNNs are publicly accessible (http://efi.igb.illinois.edu/efi-est/), so similar ‘genomic enzymology’ strategies for discovering novel pathways can be used by the community.

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Fig. 1: d-Apiose, its biological context, and an overview of the strategy for discovery of catabolic pathways for d-apiose.
Fig. 2: The nonoxidative transketolase pathway.
Fig. 3: Oxidative pathway with a xylose isomerase family decarboxylase.
Fig. 4: Oxidative pathway with an RLP decarboxylase.
Fig. 5: Oxidative pathway with an RLP transcarboxylase/hydrolase.
Fig. 6: Novel reactions and mechanisms in the catabolism of d-apiose.

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Acknowledgements

This work was supported by grants U54GM093342 (to S.C.A. and J.A.G.) and P01GM118303 (to S.C.A. and J.A.G.) from the National Institutes of Health.

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Contributions

M.S.C., X.Z., H.H., M.W.V., S.C.A., and J.A.G. conceived the project. J.T.B., M.W.V., and J.A.G. developed the library for SBP ligand screening. M.W.V., N.A., A.G., J.B.B., and S.C.A. managed the protein purification pipeline for the SBPs and some pathway enzymes. X.Z. and H.H. contributed purification of the remaining pathway enzymes, biochemical characterization of all enzymes, and chemical validation of their substrates and products. M.W.V. contributed the DSF screening of SBPs against the ligand library. M.S.C. identified d-apiose as the physiological ligand for the SBPs. M.W.V., J.B.B., and S.C.A. contributed crystallization data and analysis. M.S.C., X.Z., H.H., B.S.F., and J.A.G. evaluated SSNs, GNNs, biochemical, and biological data to hypothesize pathways. M.S.C., H.H., and R.G.Z. contributed biological validation of pathways. H.M.A. contributed molecular cloning. M.S.C., X.Z., and J.A.G. wrote the paper with contributions from all authors.

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Correspondence to John A. Gerlt.

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Supplementary Text and Figures

Supplementary Table 1–8, Supplementary Figures 1–34

Reporting Summary

Supplementary Dataset 1

Solute-binding proteins (SBPs) from PF13407 screened in this study

Supplementary Dataset 2

Phylogenetic information for organisms that encode the pathways discovered in this study. Separate worksheets are provided for each pathway

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Carter, M.S., Zhang, X., Huang, H. et al. Functional assignment of multiple catabolic pathways for d-apiose. Nat Chem Biol 14, 696–705 (2018). https://doi.org/10.1038/s41589-018-0067-7

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