A-Hydroxymuconic semialdehyde

Hydroxymorpholine. see Morpholine a-Hydroxymuconic acid, see PCB-1221 a-Hydroxymuconic semialdehyde, see Benzene... 

Dihydroxybenzoate is oxidized by extracts of Pseudomonas fluorescens with the consumption of one mole of oxygen per mole of substrate. An equivalent amount of carbon dioxide is evolved, and a-hydroxymuconic semialdehyde is formed. Intermediates between 2,3-dihydroxyben-zoate and a-hydroxymuconic semialdehyde have not been detected, nor has the site of ring cleavage been established. 

The product, a-hydroxymuconic semialdehyde, was isolated and characterized from a large scale incubation. Its elemental analysis, infrared, ultraviolet and visible absorption spectra, and its rapid decomposition to pyruvate by extracts of Pseudomonas aeruginosa T1 (JO) are all consistent with this structure. 

Intermediates between 2,3-dihydroxybenzoate and a-hydroxymuconic semialdehyde have not been detected. It is evident, however, that catechol is not an intermediate in the formation of 2,3-dihydroxybenzoate since extracts catalyze neither its formation anaerobically from 2,3-dihydroxybenzoate nor its oxidation. In fact, catechol is an inhibitor of... 

Dihydroxybenzoate was assayed at 30 °C. in a fully automated Unicam SP800 spectrophotometer at 430 m/x. The reaction cuvettes contained 0.067M phosphate buffer, pH 7.1 (2.5 ml.) 25 mM 2,3-dihydroxybenzoate (20 juliters) enzyme solution (as required but usually between 5 and 50 / liters). Under these conditions the molar extinction coefficient of a-hydroxymuconic semialdehyde at 430 mfi is 3.2 X The assays were conducted at 430 m/x because this is a more convenient wavelength when simultaneous measurements of oxygen consumption and product formation are made, although it is much less sensitive an assay than that used by Kojima et al. (5). 

The purest preparations obtained catalyze the formation of 7.3 /xmoles of a-hydroxymuconic semialdehyde/min./mg. protein after reactivation with NaBH4, anaerobically. 

Figure 2. Possible routes of a-hydroxymuconic semialdehyde formation from 2,3-dihydroxybenzoic acid...

Carboxymethyl-muconic acid 5-(y-Carboxy-y-oxo)-propenyl-4,6-dihydroxy-picolinic acid a-Hydroxymuconic semialdehyde... 

The subsequent metabolism of a-hydroxymuconic semialdehyde has been outlined by Nishizuka et al. (1962). The next step is oxidation of the aldehyde group by a dehydrogenase using DPN as electron acceptor, forming y-oxalocrotonate. Decarboxylation and hydration, catalyzed by enzymes not yet described, produce a-keto-a-hydroxyvalerate, which is oxidized in the presence of DPN, presumably to acetopyruvate, which is rapidly split to acetate and pyruvate. 

The nonenzymic reactions of the primary oxidation product in acid also support the proposed structure (Mehler, 1958). In acid solutions the amino group may be hydrolyzed to form the enol of a /3-keto acid, which may be expected to decarboxylate easily. The acid degradation product appears to be identical with a compound obtained by enzymic oxidation of catechol (see page 98). This compound has recently been isolated by Trippet et al. (1960) and characterized as a-hydroxymuconic semialdehyde. 

Riegert U, G Heiss, P Fischer, A Stolz (1998) Distal cleavage of 3-chlorocatechol by an extradiol dioxygenase to 3-chloro-2-hydroxymuconic semialdehyde. J Bacterial 180 2849-2853. 

Dioxygenases catalyze the incorporation of both atoms of molecular oxygen into the substrate. Microbial pyrocatechol dioxygenases contain non-heme iron as the sole cofactor and catalyze the oxidative cleavage of pyrocatechol to as,cis-muconic acid (intradiol equation 19) or to a-hydroxymuconic s-semialdehyde (extradiol equation 20).63"65 On... 

A spectacular example of stability enhancement through immobilization has been reported for the enzyme catechol-2,3-dioxygenase.27 This enzyme, isolated from the thermophilic bacterium Bacillus stearothermophilus, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde (which can be monitored by absorbance at 375 nm). The soluble enzyme exhibits maximal activity at 50 °C, but following immobilization on glyoxyl agarose beads with a borohydride reduction step, the optimum reaction temperature shifted to 70 °C. At a total protein concentration of 0.010 mg/mL and a temperature of 55 °C, the half-life of the soluble enzyme was 0.08 h, while the enzyme-modified beads had a half-life of 68 h. This represents a 750-fold enhancement of stability that has been attributed to the prevention of subunit dissociation upon immobilization. 

Differently, a Moraxdla strain degraded PNP by replacing the nitrogroup with a hydroxyl group and accumulating traces of hydroquinone in the medium. Hydroquinone was then converted into P-ketoadipic acid via Y hydroxymuconic semialdehyde [65]. 

For example, the enzyme pyrocatechase effects intradiol cleavage of catechol to muconic acid [72,73,74], whereas protocatechuate 3,4-dioxygenase promotes that of protocatechuic acid (R = COOH in 37) [75,76]. Metapyrocatechase is a catalyst for proximal extradiol cleavage of catechol to a-hydroxymuconic acid e-semialdehyde (38) [77] ... 

If a meta-cleavage pathway is the only one available to a particular microorganism, then the 5-chloro-2-hydroxymuconic acid semialdehyde (Eq.17-8) will accumulate unless another initial biotransformation is performed on it. 

---