Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation

Eugene Kuatsjah, Michael Zahn, Xiangyang Chen, Ryo Kato, Daniel J. Hinchen, Mikhail O. Konev, Rui Katahira, Christian Orr, Armin Wagner, Yike Zou, Stefan J. Haugen, Kelsey J. Ramirez, Joshua K. Michener, Andrew R. Pickford, Naofumi Kamimura, Eiji Masai, K. N. Houk, John E. McGeehan, Gregg T. Beckham

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

Original languageEnglish
Article numbere2212246120
JournalProceedings of the National Academy of Sciences of the United States of America
Volume120
Issue number4
DOIs
StatePublished - Jan 24 2023

Funding

ACKNOWLEDGMENTS. This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under contract no. DE-AC36-08GO28308. E.K., D.J.H., M.O.K., J.E.M., A.R.P., J.K.M., and G.T.B. acknowledge funding from the Center for Bioenergy Innovation, a U.S. Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Funding for analytical chemistry and organic synthesis was provided to S.J.H., R.K., and G.T.B. by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. government. The U.S.government retains and the publisher,by accepting the article for publication, acknowledges that the U.S. government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. government purposes. M.Z., A.R.P., and J.E.M. acknowledge Research England for Expanding Excellence in England (E3) funding. Funding to X.C. and K.N.H. was provided by the NSF (CHE-1764328). E.M. acknowledges funding from the Noda Institute for Scientific Research, Japan. This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through an Early Career Award to J.K.M. (ERKP971). Erika Erickson is thanked for the construction of pEE001 and pEE002. Nina X. Gu, Lindsay D. Eltis, and Jennifer L. DuBois are thanked for helpful discussions. We thank the Diamond Light Source (Didcot, UK) for beamtime (proposal MX-23269), the beamline staff at I23 and I03 for support, and Nikul Khunti and Diamond B21 for their help with collecting the SAXS data. Computations were performed on the Hoffman2 cluster at UCLA and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the NSF (OCI-1053575). We are grateful for financial support of the National Institute of General Medical Sciences, NIH, GM124480. Tracy Hartlage and Dr. William L. Watts, Jr from Chiral Technologies Inc. are thanked for their assistance for selection, optimization, and method development for chiral chromatography. This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under contract no. DE-AC36-08GO28308. E.K., D.J.H., M.O.K., J.E.M., A.R.P., J.K.M., and G.T.B. acknowledge funding from the Center for Bioenergy Innovation, a U.S. Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Funding for analytical chemistry and organic synthesis was provided to S.J.H., R.K., and G.T.B. by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. government. The U.S. government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. government purposes. M.Z., A.R.P., and J.E.M. acknowledge Research England for Expanding Excellence in England (E3) funding. Funding to X.C. and K.N.H. was provided by the NSF (CHE-1764328). E.M. acknowledges funding from the Noda Institute for Scientific Research, Japan. This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through an Early Career Award to J.K.M. (ERKP971). Erika Erickson is thanked for the construction of pEE001 and pEE002. Nina X. Gu, Lindsay D. Eltis, and Jennifer L. DuBois are thanked for helpful discussions. We thank the Diamond Light Source (Didcot, UK) for beamtime (proposal MX-23269), the beamline staff at I23 and I03 for support, and Nikul Khunti and Diamond B21 for their help with collecting the SAXS data. Computations were performed on the Hoffman2 cluster at UCLA and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the NSF (OCI-1053575). We are grateful for financial support of the National Institute of General Medical Sciences, NIH, GM124480. Tracy Hartlage and Dr. William L. Watts, Jr from Chiral Technologies Inc. are thanked for their assistance for selection, optimization, and method development for chiral chromatography.

Keywords

  • NTF-2
  • Novosphingobium aromaticivorans
  • Sphingobium sp. SYK-6
  • aromatic catabolism
  • lignin

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