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The Bacterial Oxidation of Vitamin B₆

VII. PURIFICATION, PROPERTIES, AND MECHANISM OF ACTION OF AN OXYGENASE WHICH CLEAVES THE 3-HYDROXYPYRIDINE RING

From the ¹ From the Department of Biochemistry, University of California, Berkeley, California 94720

Purification of an inducible oxygenase that opens the pyridine ring during the metabolic degradation of Vitamin B₆ by a soil bacterium is described. The enzyme is shown to catalyze Reaction 1, and is readily assayed by following the decrease in absorbance at 340 mµ resulting from utilization of DPNH and disappearance of the hydroxypyridine. The

[see PDF for equation]

crystalline oxygenase is homogeneous by disc gel electrophoresis and ultracentrifugation, has a sedimentation coefficient (s⁰_{20, w}) of 7.3 S and a molecular weight (by sedimentation equilibrium) of 166,000, and contains 2 moles of FAD per mole. Activity is lost on resolution with acidic ammonium sulfate and can be completely restored with FAD; FMN is inactive. Crystalline preparations of constant FAD content vary in activity from 90 to 300 arbitrary units per mg for reasons still unknown. The oxygenase is highly sensitive to oxidation and to sulfhydryl reagents, but not to chelating agents, and is stabilized by high concentrations of mercaptoethanol (0.1%) and glycerol (50%).

Under anaerobic conditions, the FAD-enzyme is reduced to FADH₂-enzyme by DPNH, NaBH₄, or Na₂S₂O₄; admission of oxygen reoxidizes the enzyme slowly, whereas on addition of oxygen and Compound I rapid reoxidation of the enzyme occurs and the open chain product appears. No spectrophotometrically detectable intermediate is observed either anaerobically or aerobically, and the available evidence indicates that the reaction proceeds in a concerted fashion via a ternary complex of oxygenase, DPNH, and Compound I. When Reaction 1 is conducted in the presence of ¹⁸O₂, the isotope is incorporated predominantly into the acetyl group, but also into the newly formed carboxyl group of the product. Thus, the enzyme is best classified as a dioxygenase, although some ¹⁸O from H₂¹⁸O also is incorporated into the product. A reaction mechanism consistent with these results is presented which also accounts for the deviation of this labeling pattern from that observed with dioxygenases which cleave aromatic rings without concomitant reduction.

TPNH is almost as effective as DPNH in Reaction 1. Of several pyridine analogues tested, only 5-pyridoxic acid replaces Compound I as a substrate; it is very much less active in this capacity. An apparently similar oxygenase from another pseudomonad cleaves 5-pyridoxic acid in preference to Compound I, and is specific for TPNH as the second substrate.

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