The metabolism of protocatechuate by Pseudomonas testosteroni

Abstract

1. Protocatechuate 4,5-oxygenase, purified 21-fold from extracts of Pseudomonas testosteroni, was examined in the ultracentrifuge and assigned a mol.wt. of about 140000. 2. When diluted, the enzyme rapidly lost activity during catalysis. Inactivation was partially prevented by l-cysteine. 3. With a saturating concentration of protocatechuate (1.36mm), K(m) for oxygen was 0.303mm. This value is greater than the concentration of oxygen in water saturated with air at 20 degrees . 4. Cell extracts converted protocatechuate into gamma-carboxy-gamma-hydroxy-alpha-oxovalerate, which was isolated as its lactone. 5. gamma-Carboxy-gamma-hydroxy-alpha-oxovalerate pyruvate-lyase activity was stimulated by Mg(2+) ions and mercaptoethanol. Cells grown with p-hydroxybenzoate as carbon source contained higher concentrations of this enzyme than those grown with succinate.

Protocatechuate 4,5-dioxygenase from Comamonas testosteroni T-2: biochemical and molecular properties of a new subgroup within class III of extradiol dioxygenases

“…Above this concentration, the reaction did not go to completion, as has been observed previously (Dagley et al 1968). We derived values of 55.5 (±2.3) lM PCA and 1.47 (±0.3) lkat for apparent K m and V max , respectively; this is close to the value (46 lM) for K m reported for C. testosteroni NCIMB 8893 (Dagley et al 1968).…”

“…Their structural fold bears no resemblance to the fold shared by class I and class II enzymes (Sugimoto et al 1999). Dagley et al (1968) cited the widespread importance of the extradiol cleavage of PCA and inferred biodiversity in the extradiol cleavage enzymes (Dagley et al 1960). PCA is the point of convergence of pathways for degradation of many aromatic compounds, for example p-toluenesulfonate in Comamonas testosteroni T-2 (Cook et al 1999), of m-chlorobenzoate in C. testosteroni BR60 (Nakatsu and Wyndham 1995), and of lignin in Sphingomonas paucimobilis SYK-6 (Noda et al 1990;Hara et al 2003).…”

Purification and characterization of protocatechuate 2,3-dioxygenase from Bacillus macerans: a new extradiol catecholic dioxygenase

“…The absorbance maxima of most of the products were observed to be 350 to 382 nm, suggesting that the substrates are cleaved in a proximal extradiol manner between C-2 and C-3. Distal extradiol cleavage would be expected to yield products with A 4w (18,19), whereas the products of intradiol cleavage would be expected to absorb in the UV range (25,65). The cleavage of homo-PCA (HPCA) and 3,4-dihydroxyphenylpropionic acid (DHPP) each gave reaction products with two absorption maxima, potentially indicating two different ring cleavage products for these compounds.…”

“…The aromatic ring of PCA is opened in reactions catalyzed by dioxygenase enzymes that result in the incorporation of both atoms of 02 into the open chain products (14,19,25,46,65). Two of these enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) (10,21,25,59,65,70) and 4,5-PCD (5,18,19,53) were among the first of this class of enzyme to be recognized. They continue to serve as prototypical enzymes for the two major subclasses of catecholic dioxygenases termed intradiol and extradiol on the basis of the site of ring cleavage (for reviews see references 37 and 46).…”

Improving alternate lignin catabolite utilization of LigAB from Sphingobium sp. strain SYK-6 through site directed mutagenesis

“…This makes LigAB the most omnivorous of the protocatechuate 4,5-dioxygenases that have been investigated to date [10][11][12][13][14][15][16]. Analysis of the LigAB crystal structure (PDB: 1B4U) shows that Phe103␣, in addition to being part of the previously identified allosteric pocket, is positioned within 5Å of the C5 position of PCA.…”