Mechanism of two flavoenzymes involved in the degradation of vitamin B6 in bacteria: MHPC oxygenase and 5-pyridoxic acid oxygenase.
Brissette, Pierre
1990
Abstract
2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase and 5-pyridoxic acid oxygenase are flavoenzymes involved in the degradation of Vitamin B$\sb6$ in bacteria. Both oxygenases have been shown to catalyze the incorporation of two atoms of oxygen into their respective substrate with concomitant cleavage of the pyridine ring of the substrate. These studies have combined spectroscopic and rapid reaction kinetic techniques to oxygen-18 tracer experiments to elucidate the oxygenation mechanism of these enzymes. Study of the reductive half-reaction of both oxygenases indicated that the rate of enzyme reduction by pyridine nucleotides was enormously stimulated in the presence of the substrate MHPC. The stimulation in enzyme reduction elicited by MHPC binding resulted from both an increased affinity of the enzyme for the pyridine nucleotides and also an increased intrinsic rate of flavin reduction. Fluorescence resonance energy transfer experiments with MHPC oxygenase suggested that MHPC lies in close proximity to the enzyme-bound FAD ($<$10A) with the plane of the pyridine ring of MHPC nearly perpendicular to the isoalloxazine ring of FAD. Such a geometry is thought to be optimal for oxygenation of the substrate by an activated flavin-oxygen adduct. Investigation of the oxidative half-reaction in the presence of the substrate, MHPC, has revealed the formation of two consecutive transient oxygenated flavin intermediates with each of these enzymes. These intermediates resemble those previously observed with the aromatic flavoprotein hydroxylases and were identified as the C(4a)-hydroperoxyflavin and C(4a)-hydroxyflavin. The C(4a)-hydroxyflavin intermediate detected in the oxidative half-reaction of MHPC oxygenase was remarkably fluorescent with a quantum yield of 0.57. Oxygen-18 tracer experiments demonstrated that the enzymatic product, AAMS, had incorporated 1 atom of oxygen from $\sp{18}$O$\sb2$ into its acetyl moiety and 0.60-0.65 atom from H$\sb2\sp{18}$O into its carboxymethyl group. Based on these observations a mechanism, similar to the hydroxylation mechanism proposed for the flavoprotein hydroxylases, was postulated for the action of MHPC and 5-pyridoxic and acid oxygenases. Conversion of the C(4a)-hydroperoxyflavin to the C(4a)-hydroxyflavin species is accompanied by the transfer of an oxygen atom to the aromatic substrate, MHPC. This hydroxylation involves an electrophilic attack of the distal oxygen of the flavin hydroperoxide on the aromatic ring of the substrate. The presence of a methyl group at the 2-position of the hydroxylated MHPC intermediate prevents rearomatization common to flavoprotein hydroxylases. Consequently, formation of the product, AAMS, arises from hydration and ring cleavage of the transient hydroxylated MHPC species.Other Identifiers
(UMI)AAI9116134
Subjects
Chemistry, Biochemistry
Types
Thesis
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