Solvent accessibility to flavin in oxynitrilase

Area of Science:

• Enzyme kinetics
• Biochemistry
• Organic chemistry

Background:

• Oxynitrilase enzymes catalyze crucial biochemical reactions.
• Flavin adenine dinucleotide (FAD) is a vital coenzyme in many enzymatic processes.
• Understanding coenzyme accessibility within enzyme active sites is key to enzyme mechanism studies.

Purpose of the Study:

• To investigate the catalytic activity and coenzyme accessibility of oxynitrilase using a modified flavin, 2-thioFAD.
• To compare the reactivity and accessibility of the 2-position of the bound coenzyme in oxynitrilase with other flavoenzymes.

Main Methods:

• Enzymatic assays using 2-thioFAD-substituted oxynitrilase.
• Spectroscopic analysis of reaction intermediates.
• Kinetic studies to determine reaction pathways.

Main Results:

• 2-thioFAD-bound oxynitrilase exhibits catalytic activity comparable to the native enzyme.
• Reactions with methyl methanethiolsulfonate and oxidants (m-chloroperoxybenzoate, H2O2) form flavin disulfide and normal flavin, respectively.
• A flavin 2-S-oxide intermediate is observed, accumulating significantly with m-chloroperoxybenzoate.
• Kinetic data indicate the 2-position of the bound coenzyme is solvent-accessible in oxynitrilase.

Conclusions:

• The 2-position of the pyrimidine ring in oxynitrilase-bound 2-thioFAD is accessible to solvent.
• This accessibility contrasts with the inaccessible 2 and 4 positions observed for flavin bound to lactate oxidase.
• The findings provide insights into the structural flexibility and active site environment of oxynitrilase.