A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOMicrobial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived a9FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2013/08293-72014/10448-12016/22956-7We acknowledge funding from NSF grants to J.L.D. (MCB-1715176), K.N.H. (CHE-1361104), and E.L.N. (DEB-1556541 and MCB-1615365) and BBSRC grants to J.E.M. (BB/P011918/1, BB/L001926/1 and a studentship to S.J.B.M.). G.T.B., M.M.M., C.W.J., M.F.C., E.L.N.,

Enabling microbial syringol conversion through structure-guided protein engineering

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds

Diverse Organization of Genes of the β-Ketoadipate Pathway in Members of the Marine Roseobacter Lineage

Members of the Roseobacter lineage, an ecologically important marine clade within the class α-Proteobacteria, harbor genes for the protocatechuate branch of the β-ketoadipate pathway, a major catabolic route for lignin-related aromatic compounds. The genes of this pathway are typically clustered, although gene order varies among organisms. Here we characterize genes linked to pcaH and -G, which encode protocatechuate 3,4-dioxygenase, in eight closely related members of the Roseobacter lineage (pairwise 16S rRNA gene sequence identities, 92 to 99%). Sequence analysis of genomic fragments revealed five unique pca gene arrangements. Identical gene organization was found for isolates demonstrating species-level identity (i.e., >99% 16S rRNA gene similarity). In one isolate, six functionally related genes were clustered: pcaQ, pobA, pcaD, pcaC, pcaH, and pcaG. The remaining seven isolates lacked at least one of these genes in their clusters, although the relative order of the remaining genes was preserved. Three genes (pcaC, -H, and -G) were physically linked in all isolates. A highly conserved open reading frame (ORF) was found immediately downstream of pcaG in all eight isolates. Reverse transcription-PCR analysis of RNA from one isolate, Silicibacter pomeroyi DSS-3, provides evidence that this ORF is coexpressed with upstream pca genes. The absence of this ORF in similar bacterial pca gene clusters from diverse microbes suggests a niche-specific role for its protein product in Roseobacter group members. Collectively, these comparisons of bacterial pca gene organization illuminate a complex evolutionary history and underscore the widespread ecological importance of the encoded β-ketoadipate pathway

Oligomerization of BenM, a LysR-type transcriptional regulator: structural basis for the aggregation of proteins in this family

A general tetramerization and oligomerization scheme is proposed for BenM and other LysR-type transcriptional regulators based on two newly characterized structures of the effector-binding domain of BenM. An analysis of these structures in the light of other LysR-type structures may explain the general solubility problems associated with this protein family