Abstract
The biological production of FDCA is of considerable value as a potential replacement for petrochemical-derived monomers such as terephthalate, used in polyethylene terephthalate (PET) plastics. HmfF belongs to an uncharacterized branch of the prenylated flavin (prFMN) dependent UbiD family of reversible (de)carboxylases and is proposed to convert 2,5-furandicarboxylic acid (FDCA) to furoic acid in vivo. We present a detailed characterization of HmfF and demonstrate that HmfF can catalyze furoic acid carboxylation at elevated CO2 levels in vitro. We report the crystal structure of a thermophilic HmfF from Pelotomaculum thermopropionicum, revealing that the active site located above the prFMN cofactor contains a furoic acid/FDCA binding site composed of residues H296-R304-R331 specific to the HmfF branch of UbiD enzymes. Variants of the latter are compromised in activity, while H296N alters the substrate preference to pyrrole compounds. Solution studies and crystal structure determination of an engineered dimeric form of the enzyme revealed an unexpected key role for a UbiD family wide conserved Leu residue in activity. The structural insights into substrate and cofactor binding provide a template for further exploitation of HmfF in the production of FDCA plastic precursors and improve our understanding of catalysis by members of the UbiD enzyme family.
Highlights
Furan-based components such as 2,5-furandicarboxylic acid are of considerable value as potentialreplacements for petrochemical-derived monomers such as terephthalate, which are used in polymers such as polyethylene terephthalate (PET) plastics.[1,2]
The pathway and associated genes for furfural and HMF degradation were first identified in the Gram-negative bacterium Cupriavidus basilensis HMF14.7 The genes are organized in two distinct clusters: HmfA−E, which are essential for both furfural and HMF degradation, and HmfF−H, which are required for HMF degradation only
There is no mention in the literature regarding the ability of G. kaustophilus to degrade HMF, a C. basilensis HmfF homologue (WP_011229502) could be located on pHTA426, possessing 51% sequence identity and located adjacent to a HmfG/UbiX homologue
Summary
Holo-PtHmfF was obtained by reconstituting singly expressed HmfF with reduced prFMN under anaerobic conditions as described previously.[19] Reaction of a mixture consisting of 1 mM FMN, 2 mM DMAP (Sigma), 50 μM Fre reductase, and 50 μM UbiX in buffer A was started by the addition of 5 mM NADH. Crystallographic table of data collection and refinement statistics, phylogenetic tree of UbiD/Fdc[1] family decarboxylases, production of soluble and active HmfF, PtHmfF enzyme stability, circular dichroism spectra of PtHmfF, HmfF H/D exchange monitored by NMR, electron density for bound cofactor and metal ions in PtHmF, overlay of crystal structures of UbiD enzyme family members, and UV−vis spectra obtained for the P. thermopropionicum HmfF dimer mutant (PDF). Payne: 0000-0002-6331-6374 Cunyu Yan: 0000-0002-3603-2421 Igor Larrosa: 0000-0002-5391-7424 David Leys: 0000-0003-4845-8443
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