Characterization of an extradiol dioxygenase involved in the catabolism of lignin‐derived biphenyl

Kuatsjah, Eugene; Chen, Hongming; Withers, Stephen G.; Eltis, Lindsay D.

doi:10.1002/1873-3468.12611

Cited by 23 publications

(20 citation statements)

References 31 publications

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“…To identify the LigY reaction products, OH-DDVA was incubated with a mixture of LigZ and LigY (potassium phosphate (I ϭ 0.1 M), pH 7.5). This approach minimized the non-enzymatic transformation of DCHM-HOPDA, the LigZ-produced MCP (8). HPLC analysis of the reaction products revealed the presence of 5CVA, consistent with an earlier report (9), together with another major peak (retention time (t R ) ϭ 8 min), the amount of which was proportional to 5CVA (Fig.…”

Section: Ligy Hydrolyzes Dchm-hopda To 5cva and Chpdsupporting

confidence: 90%

“…The demethylation of DDVA by a ligX-encoded oxygenase yields 2,2Ј,3-trihydroxy-3Ј-methoxy-5,5Ј-dicarboxybiphenyl (OH-DDVA) (6). meta-Cleavage of OH-DDVA by LigZ, an extradiol dioxygenase (7), yields 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (DCHM-HOPDA) (8). DCHM-HOPDA is then transformed by LigY, a proposed meta-cleavage product (MCP) hydrolase, to yield 5-carboxyvanillate (5CVA) and a second product that was presumed to be 4-carboxy-2-hydroxypenta-2,4-dienoate (CHPD) but was not identified (9).…”

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confidence: 99%

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The bacterial meta-cleavage hydrolase LigY belongs to the amidohydrolase superfamily, not to the α/β-hydrolase superfamily

Kuatsjah¹,

Chan

Kobylarz

et al. 2017

Journal of Biological Chemistry

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Strain SYK-6 of the bacterium sp. catabolizes lignin-derived biphenyl via a-cleavage pathway. In this pathway, LigY is proposed to catalyze the hydrolysis of the -cleavage product (MCP) 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate. Here, we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2,4-dienoate as the products and determined the and / values as 9.3 ± 0.6 s and 2.5 ± 0.2 × 10 m s, respectively. Sequence analyses and a 1.9 Å resolution crystal structure established that LigY belongs to the amidohydrolase superfamily, unlike previously characterized MCP hydrolases, which are serine-dependent enzymes of the α/β-hydrolase superfamily. The active-site architecture of LigY resembled that of α-amino-β-carboxymuconic-ϵ-semialdehyde decarboxylase, a class III amidohydrolase, with a single zinc ion coordinated by His-6, His-8, His-179, and Glu-282. Interestingly, we found that LigY lacks the acidic residue proposed to activate water for hydrolysis in other class III amidohydrolases. Moreover, substitution of His-223, a conserved residue proposed to activate water in other amidohydrolases, reduced the to a much lesser extent than what has been reported for other amidohydrolases, suggesting that His-223 has a different role in LigY. Substitution of Arg-72, Tyr-190, Arg-234, or Glu-282 reduced LigY activity over 100-fold. On the basis of these results, we propose a catalytic mechanism involving substrate tautomerization, substrate-assisted activation of water for hydrolysis, and formation of a-diol intermediate. This last step diverges from what occurs in serine-dependent MCP hydrolases. This study provides insight into C-C-hydrolyzing enzymes and expands the known range of reactions catalyzed by the amidohydrolase superfamily.

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Section: Ligy Hydrolyzes Dchm-hopda To 5cva and Chpdsupporting

confidence: 90%

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confidence: 99%

The bacterial meta-cleavage hydrolase LigY belongs to the amidohydrolase superfamily, not to the α/β-hydrolase superfamily

Kuatsjah¹,

Chan

Kobylarz

et al. 2017

Journal of Biological Chemistry

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“…4‐Me‐HOPDA rapidly converted to a colorless species ( t 1/2 ≈4 s) under these conditions. The dienolate form of 4‐Me‐HOPDA could be regenerated under alkaline pH, as reported for DCHM‐HOPDA …”

Section: Resultsmentioning

confidence: 63%

“…Overall, methyl substitution of the dienol moiety increased the p K a value of HOPDA. The corresponding chlorinated HOPDAs had lower p K a values, consistent with the greater electronegativity of the chloro substituent …”

Section: Resultsmentioning

confidence: 76%

Bacterial Catabolism of Biphenyls: Synthesis and Evaluation of Analogues

et al. 2018

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A series of alkylated 2,3-dihydroxybiphenyls has been prepared on the gram scale by using an effective Directed ortho Metalation-Suzuki-Miyaura cross-coupling strategy. These compounds have been used to investigate the substrate specificity of the meta-cleavage dioxygenase BphC, a key enzyme in the microbial catabolism of biphenyl. Isolation and characterization of the meta-cleavage products will allow further study of related processes, including the catabolism of lignin-derived biphenyls.

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“…DDVA is O demethylated by DDVA O-demethylase, which consists of an oxygenase component (LigXa), ferredoxin (LigXc), and ferredoxin reductase (LigXd), to generate 2,2=,3-trihydroxy-3=-methoxy-5,5=-dicarboxybiphenyl (OH-DDVA) (11,12). One of the two aromatic rings of OH-DDVA is cleaved by OH-DDVA dioxygenase (LigZ) (13,14), and the resulting meta-cleavage compound is hydrolyzed to 5-carboxyvanillate (5CVA) and 4-carboxy-2-hydroxypenta-2,4-dienoate (CHPD) by LigY (13,15). 5CVA is further converted to vanillate by 5CVA decarboxylases (LigW and LigW2) (16,17), and vanillate then enters the tricarboxylic acid (TCA) cycle via O demethylation and the protocatechuate 4,5-cleavage pathway (10,18).…”

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confidence: 99%

DdvK, a Novel Major Facilitator Superfamily Transporter Essential for 5,5′-Dehydrodivanillate Uptake by Sphingobium sp. Strain SYK-6

Mori

Niinuma

Fujita

et al. 2018

Appl Environ Microbiol

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The microbial conversion of lignin-derived aromatics is a promising strategy for the industrial utilization of this large biomass resource. However, efficient application requires an elucidation of the relevant transport and catabolic pathways. In sp. strain SYK-6, most of the enzyme genes involved in 5,5'-dehydrodivanillate (DDVA) catabolism have been characterized, but the transporter has not yet been identified. Here, we identified SLG_07710 () and SLG_07780 (), genes encoding a putative major facilitator superfamily (MFS) transporter and MarR-type transcriptional regulator, respectively. A mutant of SYK-6 completely lost the capacity to grow on and convert DDVA. DdvR repressed the expression of the DDVA-demethylase oxygenase component gene (), while DDVA acted as the gene inducer. A DDVA uptake assay was developed by employing this DdvR-controlled transcriptional regulatory system. A UT26S transformant expressing acquired DDVA uptake capacity, indicating that encodes the DDVA transporter. DdvK, probably requiring the proton motive force, was suggested to be a novel MFS transporter on the basis of the amino acid sequence similarity. Subsequently, we evaluated the effects of overexpression on the production of the DDVA metabolite 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers. A SYK-6 mutant of the PDC hydrolase gene () cultured in DDVA accumulated PDC via 5-carboxyvanillate and grew by utilizing 4-carboxy-2-hydroxypenta-2,4-dienoate. The introduction of a -expression plasmid into a mutant increased the growth rate in DDVA and the amounts of DDVA converted and PDC produced after 48 h by 1.35- and 1.34-fold, respectively. These results indicate that enhanced transporter gene expression can improve metabolite production from lignin derivatives. The bioengineering of bacteria to selectively transport and metabolize natural substrates into specific metabolites is a valuable strategy for industrial-scale chemical production. The uptake of many substrates into cells requires specific transport systems, and so the identification and characterization of transporter genes are essential for industrial applications. A number of bacterial major facilitator superfamily transporters of aromatic acids have been identified and characterized, but many transporters of lignin-derived aromatic acids remain unidentified. The efficient conversion of lignin, an abundant but unutilized aromatic biomass resource, to value-added metabolites using microbial catabolism requires the characterization of transporters for lignin-derived aromatics. In this study, we identified the transporter gene responsible for the uptake of 5,5'-dehydrodivanillate, a lignin-derived biphenyl compound, in sp. strain SYK-6. In addition to characterizing its function, we applied this transporter gene to the production of a value-added metabolite from 5,5'-dehydrodivanillate.

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Characterization of an extradiol dioxygenase involved in the catabolism of lignin‐derived biphenyl

Cited by 23 publications

References 31 publications

The bacterial meta-cleavage hydrolase LigY belongs to the amidohydrolase superfamily, not to the α/β-hydrolase superfamily

The bacterial meta-cleavage hydrolase LigY belongs to the amidohydrolase superfamily, not to the α/β-hydrolase superfamily

Bacterial Catabolism of Biphenyls: Synthesis and Evaluation of Analogues

DdvK, a Novel Major Facilitator Superfamily Transporter Essential for 5,5′-Dehydrodivanillate Uptake by Sphingobium sp. Strain SYK-6

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