Douglas W. Ribbons

3-Hydroxybenzoate 6-hydroxylase from Pseudomonas aeruginosa

Abstract An inducible 3-hydroxybenzoate 6-hydroxylase has been purified to homogeneity from Pseudomonas aeruginosa . It contains FAD as a prosthetic group. 3-Hydroxybenzoate is quantitatively hydroxylated to give gentisate with equimolar consumptions of NADH and O2. NADPH will substitute as an electron donor, and several aromatic analogues of 3-hydroxybenzoate stimulate reduced nucleotide oxidation by the enzyme with formation of both hydrogen peroxide and hydroxylated products. Of various analogues of 3-hydroxybenzoate, those substituted in 2,4,5 and 6-positions are competent substrates; partial uncoupling of electron flow from hydroxylation with concomitant formation of hydrogen peroxide and “gentisates” occurs. The “natural” product of the reaction, gentisate, is an effector in that it stimulates NADH oxidation with the formation of hydrogen peroxide. 3-hydroxybenzoate 6-hydroxylase thus resembles other flavoprotein hydroxylases in the general regulatory properties dictated by their aromatic substrates, pseudosubstrates or effectors.

3-Hydroxybenzoate 4-hydroxylase from Pseudomonas testosteroni

Summary 3-Hydroxybenzoate 4-hydroxylase has been purified to homogeneity from extracts from extracts pf Ps . testosteroni . It is a flavoprotein (FAD) which catalyzes the transformation of 3-hydroxybenzoate to protocatechuate with equimolar consumption of NADPH and O 2 . NADH is a poor substitute for NADPH. Several analogues of 3-hydroxybenzoate substituted in the 2,4,5 and 6 positions, act as effectors and substrates for NADPH oxidation but with varying efficiencies of hydroxylation. 2,3-, 2,5-, 3,5-dihydroxybenzoates, 3-hydroxyanthranilate, 2-fluoro-5-hydroxybenzoate and 4-fluoro-3-hydroxybenzoate are competent substrates.

Specificity of monohydric phenol oxidations by meta cleavage pathways in Pseudomonas aeruginosa T1

SummaryThe oxidation of several mono-hydric phenols by wild type and mutant strains of Pseudomonas aeruginosa T1 has been studied. The data suggest, that a non-specific enzyme sequence of the meta cleavage pathway is induced by all of these phenols, which can catalyze the oxidation of phenol and its analogues to pyruvate, a fatty acid and a carbonyl compound, according to the general scheme of Dagley et al. (1964). Mutants unable to grow on phenol (hydroxylase-negative), have been isolated, and they are also unable to grown on or oxidize the cresols and the xylenols. Revertants of these mutants regain the capacity to grow on all these phenols and are indistinguishable from the wild type. Induced-substrate relationships for the earlier enzymes of the pathway have been determined, e.g., phenol in addition to catechol and the methylcatechols is an inducer for catechol 2,3-oxygenase. Analysis of the enzymic content of cells grown in a variety of steadystate conditions shows (a) that the ratio of the specific activities of the “phenol” hydroxylase and catechol 2,3-oxygenase is constant for each of their analogous substrates; and (b) that induction and catabolite repression of catechol 2,3-oxygenase and the “muconic semialdehyde” hydrolyase are coordinate, but that control of the “phenol” hydroxylase is independent.

Fine structure of Methanomonas methanooxidans

SummaryThin sections of the obligate methane oxidizing bacterium, Methanomonas methanooxidans, reveal extensive intracytoplasmic membranes. These always occur in a peripheral arrangement. They appear similar to those in other bacteria which utilize specific energy sources. Their structure and function is discussed.

Utilization of phthalate esters by micrococci

Several strains of Micrococcus have been isolated by enrichment with one of several phthalate esters as sole carbon source. They have been separated into four groups by their esterase content and nutritional characteristics. The catabolic potential for phthalate utilization found in these strains provides further support for designation of the four groups. Pathways for phthalate utilization by 4,5-dihydroxyphthalate and/or 3,4-dihydroxyphthalate and protocatechuate and/or 2,3-dihydroxybenzoate are outlined, which suggests that micrococci possess substantial potential for the catabolism of aromatic compounds.

2,3-Dihydroxybenzoate 3,4-oxygenase from Pseudomonas fluorescens—Oxidation of a substrate analog

2,3-Dihydroxybenzoate is oxidized by extracts of Pseudomonas fluorescens to α-hydroxymuconic semialdehyde and CO2. A noninducing substrate analog, 2,3-dihydroxy-p-toluate, has now been used to determine the site of ring cleavage. 2,3-Dihydroxy-p-toluate is oxidized quantitatively with the consumption of 1 mole of O2, evolution of 1 mole of CO2, and the accumulation of 2,6-diketoheptenoate. The product was characterized by (1) spectral analysis of the isolated acid, (2) ring closure in the presence of NH4+ to form 6-methylpicolinate, and (3) its ready conversion to acetate in 76% yield, by extracts of Ps. aeruginosa T1. It is concluded that enzymic cleavage of the benzenoid nucleus by this oxygenase is between carbon atoms 3 and 4, and the enzyme has been named 2,3-dihydroxybenzoate 3,4-oxygenase.

The transformation of phthalaldehydate by phthalate-grown Micrococcus strain 12B.

Abstract Micrococcus strain 12B, grown with phthalate, transformed the phthalate analog, phthalaldehydate (2-formylbenzoate), to 3,4-dihydroxyphthalaldehydate which was isolated and identified as its lactol. An 18 O 2 incorporation experiment indicated that a dioxygenase mechanism was involved. It is proposed by analogy, that phthalate is metabolized through cis -3,4-dihydro-3,4-dihydroxyphthalate and 3,4-dihydroxyphthalate by this bacterium.