3270-98-2

Usage

Definition

ChEBI: A 2-hydroxy-6-oxohexa-2,4-dienoic acid having (2E,4Z)-configuration.

InChI:InChI=1/C6H6O4/c7-4-2-1-3-5(8)6(9)10/h1-4,7H,(H,9,10)/b3-1+,4-2+

Upstream product

• 120-80-9 benzene-1,2-diol 
• 672-67-3 2H-pyran-2-one-6-carboxylic acid 
• 16597-58-3 2-amino-3-carboxymuconic acid 6-semialdehyde 

Downstream Products

• 64-18-6 formic acid 
• 50480-68-7 2-hydroxy-2,4-pentadienoic acid 

Relevant academic research and scientific papers

High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions

Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of L-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction. Catechol released from TPL-catalyzed cleavage of 3,4-dihydroxy-L-phenylalanine (L-DOPA) was further oxidized by C23O to form 2-hydroxymuconate semialdehyde, which could be readily detected by spectrophotometric measurements at 375 nm. The assay achieved a unique balance between the ease of operation and superiority of analytical performances including linearity, sensitivity and accuracy. In addition, this assay enabled real-time monitoring of TPL activity with high efficiency and reliability. As C23O is highly specific towards catechol, a non-natural product of microorganism, the assay was therefore accessible to both crude cell extracts and the whole-cell system without elaborate purification steps of enzymes, which could greatly expedite discovery and engineering of TPLs. This study provided fundamental principle for high-throughput screening of other enzymes consuming or producing catechol derivatives.

Microbial utilization of lignin-derived aromatics: Via a synthetic catechol meta -cleavage pathway

Lignin is an abundant organic reservoir from plant biomass that is underexploited. Microbial utilization of lignin represents a sustainable approach for a biorefinery. In this study, we diversified the bioproduction from lignin-degraded aromatic compounds based on a catechol meta-cleavage metabolism. To establish an efficient downstream ligninolytic pathway, debottlenecking of the rate-limiting protocatechuate decarboxylation and evaluation of the alternative meta-cleaving pathways at the enzymatic and metabolic levels have been conducted. As a carbon flux indicator and showcase of versatility, we devised a new synthetic route towards 1,3-BDO, resulting in 0.343 g g-1 product accumulation from protocatechuate. We further extended the pathway to successfully assimilate commonly present depolymerized lignin monomers, isoeugenol, vanillin and vanillate, to central metabolites pyruvate and acetyl-CoA, as demonstrated by biocatalysis of up to 0.153 g g-1 citramalate from these aromatic monomers. The established synthetic ligninolysis via catechol meta-cleavage can serve as an efficient platform for expanded spectrum for lignin utilization.

Expression, purification, characterization and in silico analysis of newly isolated hydrocarbon degrading bleomycin resistance dioxygenase

In the present investigation, we report cloning, expression, purification and characterization of a novel Bleomycin Resistance Dioxygenase (BRPD). His-tagged fusion protein was purified to homogeneity using Ni-NTA affinity chromatography, yielding 1.2 mg of BRPD with specific activity of 6.25 U mg?1 from 600 ml of E. coli culture. Purified enzyme was a dimer with molecular weight ~ 26 kDa in SDS-PAGE and ~ 73 kDa in native PAGE analysis. The protein catalyzed breakdown of hydrocarbon substrates, including catechol and hydroquinone, in the presence of metal ions, as characterized via spectrophotometric analysis of the enzymatic reactions. Bleomycin binding was proven using the EMSA gel retardation assay, and the putative bleomycin binding site was further determined by in silico analysis. Molecular dynamic simulations revealed that BRPD attains octahedral configuration in the presence of Fe2+ ion, forming six co-ordinate complexes to degrade hydroquinone-like molecules. In contrary, in the presence of Zn2+ ion BRPD adopts tetrahedral configuration, which enables degradation of catechol-like molecules.

Reassignment of the human aldehyde dehydrogenase ALDH8A1 (ALDH12) to the kynurenine pathway in tryptophan catabolism

The kynurenine pathway is the primary route for L-tryptophan degradation in mammals. Intermediates and side products of this pathway are involved in immune response and neurode-generative diseases. This makes the study of enzymes, especially those from mammalian sources, of the kynurenine pathway worthwhile. Recent studies on a bacterial version of an enzyme of this pathway, 2-aminomuconate semialdehyde (2-AMS) dehydrogenase (AMSDH), have provided a detailed understanding of the catalytic mechanism and identified residues conserved for muconate semialdehyde recognition and activation. Findings from the bacterial enzyme have prompted the reconsideration of the function of a previously identified human aldehyde dehydrogenase, ALDH8A1 (or ALDH12), which was annotated as a retinal dehydrogenase based on its ability to preferentially oxidize 9-cis-retinal over trans-retinal. Here, we provide compelling bioinformatics and experimental evidence that human ALDH8A1 should be reassigned to the missing 2-AMS dehydrogenase of the kynurenine metabolic pathway. For the first time, the product of the semialdehyde oxidation by AMSDH is also revealed by NMR and high-resolution MS. We found that ALDH8A1 catalyzes the NAD-dependent oxidation of 2-AMS with a catalytic efficiency equivalent to that of AMSDH from the bacterium Pseudomonas fluorescens. Substitution of active-site residues required for substrate recognition, binding, and isomerization in the bacterial enzyme resulted in human ALDH8A1 variants with 160-fold increased Km or no detectable activity. In conclusion, this molecular study establishes an additional enzymatic step in an important human pathway for tryptophan catabolism.

Single Turnover Reveals Oxygenated Intermediates in Toluene/o-Xylene Monooxygenase in the Presence of the Native Redox Partners

Toluene/o-xylene monooxygenase (ToMO) is a non-heme diiron protein that activates O2 for subsequent arene oxidation. ToMO utilizes four protein components, a catalytic hydroxylase, a regulatory protein, a Rieske protein, and a reductase. O2 activation and substrate hydroxylation in the presence of all four protein components is examined. These studies demonstrate the importance of native reductants by revealing reactivity unobserved when dithionite and mediators are used as the reductant. This reactivity is compared with that of other O2-activating diiron enzymes.