Maize microsomal benzoxazinone N-monooxygenase

Area of Science:

• Plant Biochemistry
• Enzymology
• Molecular Biology

Background:

• Benzoxazinones exist in maize (Zea mays L.) as hydroxamic acids (e.g., DIBOA) and lactam forms (e.g., HBOA).
• Hydroxamic forms are more abundant than lactam forms in maize tissue.
• DIBOA is 2,4-dihydroxy-1,4-benzoxazin-3-one, and its lactam counterpart is HBOA (2-hydroxy-1,4-benzoxazin-3-one).

Purpose of the Study:

• To identify and characterize the enzyme responsible for N-hydroxylation of HBOA to DIBOA in maize.
• To elucidate the biochemical properties and potential cytochrome P-450 dependence of this maize enzyme.

Main Methods:

• Enzyme assays using maize microsomal preparations.
• Investigated enzyme activity, cofactor requirements (NADPH), and inhibition by various reagents (sulfhydryl reagents, CO, N2).
• Determined kinetic parameters (K(m)), pH and temperature optima, and spectral analysis (CO-reduced difference spectrum).

Main Results:

• An enzyme catalyzing HBOA N-hydroxylation to DIBOA was identified in maize seedlings.
• The enzyme requires NADPH, exhibits optimal activity at pH 7.5 and 35°C, and is sensitive to CO and N2.
• CO-reduced difference spectra showed a 450 nm peak, supporting a cytochrome P-450 mechanism.

Conclusions:

• The identified enzyme is a NADPH-dependent N-monooxygenase, likely a cytochrome P-450.
• This enzyme plays a crucial role in the biosynthesis of benzoxazinone hydroxamic acids in maize.
• Enzyme activity coincides with the onset of hydroxamic acid accumulation in maize seedlings.