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

DOI: 10.1073/pnas.2212246120

Authors: Eugene Kuatsjah (National Renewable Energy Laboratory (USA)) , Michael Zahn (University of Portsmouth) , Xiangyang Chen (University of California Los Angeles) , Ryo Kato (Nagaoka University) , Daniel J. Hinchen (University of Portsmouth) , Mikhail O. Konev (National Renewable Energy Laboratory (USA)) , Rui Katahira (National Renewable Energy Laboratory (USA)) , Christian Orr (Diamond Light Source) , Armin Wagner (Diamond Light Source) , Yike Zou (University of California Los Angeles) , Stefan J. Haugen (National Renewable Energy Laboratory (USA)) , Kelsey J. Ramirez (National Renewable Energy Laboratory (USA)) , Joshua K. Michener (Oak Ridge National Laboratory) , Andrew R. Pickford (University of Portsmouth) , Naofumi Kamimura (Nagaoka University of Technology) , Eiji Masai (Nagaoka University of Technology) , Kendall N. Houk (University of California Los Angeles) , John Mcgeehan (University of Portsmouth) , Gregg T. Beckham (National Renewable Energy Laboratory (USA))
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences , VOL 120

State: Published (Approved)
Published: January 2023
Diamond Proposal Number(s): 23269

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.

Journal Keywords: lignin; aromatic catabolism; Novosphingobium aromaticivorans; Sphingobium sp. SYK-6; NTF-2

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: B21-High Throughput SAXS , I03-Macromolecular Crystallography , I23-Long wavelength MX

Added On: 23/01/2023 08:18

Documents:
pnas.2212246120.pdf

Discipline Tags:

Biotechnology Biochemistry Catalysis Chemistry Structural biology Engineering & Technology Life Sciences & Biotech

Technical Tags:

Diffraction Scattering Macromolecular Crystallography (MX) Long Wavelength Crystallography Small Angle X-ray Scattering (SAXS)