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A pyridoxal phosphate–dependent enzyme that oxidizes an unactivated carbon-carbon bond

Nature Chemical Biology volume 12, pages 194–199 (2016)Cite this article

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Abstract

Pyridoxal 5′-phosphate (PLP)-dependent enzymes have wide catalytic versatility but are rarely known for their ability to react with oxygen to catalyze challenging reactions. Here, using in vitro reconstitution and kinetic analysis, we report that the indolmycin biosynthetic enzyme Ind4, from Streptomyces griseus ATCC 12648, is an unprecedented O2- and PLP-dependent enzyme that carries out a four-electron oxidation of L-arginine, including oxidation of an unactivated carbon-carbon (C-C) bond. We show that the conjugated product of this reaction, which is susceptible to nonenzymatic deamination, is efficiently intercepted and stereospecifically reduced by the partner enzyme Ind5 to give D-4,5-dehydroarginine. Thus, Ind4 couples the redox potential of O2 with the ability of PLP to stabilize anions to efficiently oxidize an unactivated C-C bond, with the subsequent stereochemical inversion by Ind5 preventing off-pathway reactions. Altogether, these results expand our knowledge of the catalytic versatility of PLP-dependent enzymes and enrich the toolbox for oxidative biocatalysis.

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Figure 1: Biosynthetic pathway of indolmycin based on previous work.
Figure 2: Ind4-catalyzed oxidation of L-arginine.
Figure 3: Ind4 and Ind5–Ind6 catalyze the construction of 1.
Figure 4: The Ind4- and Ind5–Ind6-coupled reaction catalyzes the production of 1.
Figure 5: Transient kinetic analysis of the reaction of Ind4 with L-arginine.

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Acknowledgements

We are grateful to O. Li (Zhejiang Sci-Tech University) for the gift of genomic DNA of Paenibacillus elgii, to M. Tanner for helpful discussions and use of equipment for anaerobic work, and to B. Moore for feedback on the manuscript. This work is supported by grants from Genome British Columbia (SOF148, to K.S.R.) and Natural Sciences and Engineering Research Council of Canada (402631-2011, to K.S.R., and 171359-13, to L.D.E.). Y.-L.D. was supported by a Michael Smith Foundation for Health Research Trainee Award, K.S.R. is supported by a Canadian Institutes of Health Research New Investigator Award, and L.D.E. holds a Tier 1 Canada Research Chair.

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Authors and Affiliations

  1. Department of Chemistry, The University of British Columbia, Vancouver, British Columbia, Canada

    Yi-Ling Du, Lona M Alkhalaf, Hai-Yan He & Katherine S Ryan

  2. Department of Microbiology and Immunology, The University of British Columbia, Vancouver, British Columbia, Canada

    Rahul Singh & Lindsay D Eltis

  3. Genome Science and Technology Program, The University of British Columbia, Vancouver, British Columbia, Canada

    Eugene Kuatsjah

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  1. Yi-Ling Du
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Contributions

Y.-L.D. and K.S.R. designed the project, analyzed data, and wrote the manuscript. Y.-L.D. performed the majority of experimental work. R.S., E.K. and L.D.E. performed and analyzed kinetic and spectroscopic experiments and contributed to manuscript writing. L.M.A. characterized compounds and contributed to data analysis and manuscript writing, and H.-Y.H. contributed to experimental design.

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Correspondence to Katherine S Ryan.

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Supplementary Results and Supplementary Figures 1–16. (PDF 11578 kb)

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Du, YL., Singh, R., Alkhalaf, L. et al. A pyridoxal phosphate–dependent enzyme that oxidizes an unactivated carbon-carbon bond. Nat Chem Biol 12, 194–199 (2016). https://doi.org/10.1038/nchembio.2009

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