Abstract:Sphingobium sp. strain SYK-6 is able to use a phenylcoumaran-type biaryl, dehydrodiconiferyl alcohol (DCA), as a sole source of carbon and energy. In SYK-6 cells, the alcohol group of the B-ring side chain of DCA was first oxidized to the carboxyl group, and then the alcohol group of the A-ring side chain was oxidized to generate 5-(2-carboxyvinyl)-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylate (DCA-CC). We identified phcF, phcG and phcH, which conferred the ability to convert DCA-… Show more
“…HPLC-based assays further revealed that the LSD4-catalyzed cleavage of DCA-S and lignostilbene yielded 1.1 ± 0.3 mol and 2.6 ± 0.2 mol vanillin per mol O 2 consumed, respectively. Overall, these analyses demonstrated that LSD4 catalyzes the cleavage of DCA-S to vanillin and 5-formylferulate, consistent with the proposed DCA catabolic pathway of SYK-6 ( 14 , 16 ), and that the cleavage of lignostilbene and DCA-S is well coupled to O 2 consumption. Figure 4 LSD4-catalyzed DCA-S cleavage products.…”
Section: Resultssupporting
confidence: 69%
“…By contrast, the enzymes that oxidize the methyl alcohol attached to the coumaran moiety of DCA-C have been partially characterized. PhcC, PhcD, and an unidentified dehydrogenase catalyze the oxidation of DCA-C to the two stereoisomers of 5-(2-carboxyvinyl)-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylate (DCA-CC) via an aldehyde intermediate, 3-(3-formyl-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)acrylate (DCA-CL) ( 16 ). PhcC and PhcD are glucose-methanol-choline oxidoreductases, and their reactions are coupled to respiration ( 15 ).…”
mentioning
confidence: 99%
“…PhcC and PhcD are glucose-methanol-choline oxidoreductases, and their reactions are coupled to respiration ( 15 ). The two stereoisomers of DCA-CC are decarboxylated to form DCA-S in a reaction involving PhcF, PhcG, and, to some extent, PhcH ( 16 ). Figure 1 Proposed catabolism of DCA in SYK-6.…”
Lignostilbene-α,β-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes.
Sphingobium
sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from β-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼10
6
M
−1
s
−1
) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.
“…HPLC-based assays further revealed that the LSD4-catalyzed cleavage of DCA-S and lignostilbene yielded 1.1 ± 0.3 mol and 2.6 ± 0.2 mol vanillin per mol O 2 consumed, respectively. Overall, these analyses demonstrated that LSD4 catalyzes the cleavage of DCA-S to vanillin and 5-formylferulate, consistent with the proposed DCA catabolic pathway of SYK-6 ( 14 , 16 ), and that the cleavage of lignostilbene and DCA-S is well coupled to O 2 consumption. Figure 4 LSD4-catalyzed DCA-S cleavage products.…”
Section: Resultssupporting
confidence: 69%
“…By contrast, the enzymes that oxidize the methyl alcohol attached to the coumaran moiety of DCA-C have been partially characterized. PhcC, PhcD, and an unidentified dehydrogenase catalyze the oxidation of DCA-C to the two stereoisomers of 5-(2-carboxyvinyl)-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylate (DCA-CC) via an aldehyde intermediate, 3-(3-formyl-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)acrylate (DCA-CL) ( 16 ). PhcC and PhcD are glucose-methanol-choline oxidoreductases, and their reactions are coupled to respiration ( 15 ).…”
mentioning
confidence: 99%
“…PhcC and PhcD are glucose-methanol-choline oxidoreductases, and their reactions are coupled to respiration ( 15 ). The two stereoisomers of DCA-CC are decarboxylated to form DCA-S in a reaction involving PhcF, PhcG, and, to some extent, PhcH ( 16 ). Figure 1 Proposed catabolism of DCA in SYK-6.…”
Lignostilbene-α,β-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes.
Sphingobium
sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from β-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼10
6
M
−1
s
−1
) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.
“…Lignostilbenoids occur in a very limited number of naturally occurring lignins but are prevalent in industrial lignins due to condensation reactions (10,11). Moreover, lignin-derived stilbenes are proposed to be produced in the bacterial catabolism of diaryl propane and phenylcoumarane (7,12). Plants produce other stilbenes, such as resveratrol, arachidins, and astringins, as allelochemicals to protect against pathogens and physical damage (13).…”
Section: Lignostilbene-␣-dioxygenase a (Lsda) From The Bacteriummentioning
confidence: 99%
“…Plants produce other stilbenes, such as resveratrol, arachidins, and astringins, as allelochemicals to protect against pathogens and physical damage (13). This variety of stilbenes may explain why some bacterial strains contain multiple LSD homologs (5,7,12,14,15). The first LSD to be described, LsdA from Sphingomonas paucimobilis TMY1009 (TMY1009 hereafter), shares 98% amino acid sequence identity with LSD1 from Sphingobium sp.…”
Section: Lignostilbene-␣-dioxygenase a (Lsda) From The Bacteriummentioning
Bioconversion is being regarded as a promising way for lignin valorization because it enables funneling diverse lignin components into single compounds, overcoming the heterogeneity of lignin. Although numerous lignin‐derived aromatic monomers have been funneled to target compounds in previous studies, the bioconversion of low‐molecular‐weight lignin (LMW‐lignin) fragments, for example, lignin‐derived dimers, has been rarely systematically studied, impeding further conversion of lignin. In this study, coculture systems were designed and developed to funnel multiple lignin‐derived dimers to cis, cis‐muconate and gallate by combining lignin‐derived dimers cleavage bacterium Sphingobium sp. and monomers conversion bacterium Rhodococcus opacus. With the developed coculture systems, β‐O‐4 type dimer guaiacylglycerol‐β‐guaiacyl ether, 4‐O‐5 type dimer 4,4′‐dihydroxydiphenyl ether, β‐5 type dimer balanophonin, β‐β type dimer pinoresinol, β‐1 type dimer 1,2‐bis(4‐hydroxy‐3‐methoxyphehyl)‐1,3‐propanediol and 5‐5 type dimer 5,5′‐dehydrodivanillate were converted to cis, cis‐muconate. Additionally, the developed coculture systems also showed potential in conversion of lignin‐derived dimers to gallate. The application of alkali lignin for cis, cis‐muconate production further demonstrated the effectiveness of the designed coculture systems. Overall, the developed coculture systems are beneficial to lignin biological valorization, and also provide references for the valorization of other bio‐resources.
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