Isobutylamine N-hydroxylase (IBAH) and flavin reductase (FRED) from Streptomyces viridifaciens are part of a two-component flavin-dependent monooxygenase enzyme system that catalyze the conversion of isobutylamine (IBA) to isobutylhydroxylamine (IBHA), a key step in the formation of valanimycin, an azoxy antibiotic. In this work, we present the over-expression, purification and biochemical characterization of this two-component enzyme system. IBAH and FRED were expressed and purified to homogeneity as separate proteins. FRED exhibited the oxidoreductase activity by catalyzing the oxidation of NADPH. The hydroxylation activity of IBAH was confirmed using liquid chromatography – mass spectrometry (LC-MS). Steady state kinetic data showed an oxidation activity of the monooxygenase component which proceeded at 1.97 ± 0.06 s⁻¹ as measured from oxygen consumption and in product formation, the rate was 0.012 ± 0.001 s⁻¹ , suggesting a high degree of uncoupling between product formation and oxygen consumption. In pre-steady state kinetic characterization studies, the FRED-catalyzed reduction of FAD by NADPH occurred at a rate of 10.0 ± 0.2 s⁻¹ and the KM was 490 ± 40 µM. The rate of reduction was ~1.5-fold decreased in the presence of substrate IBA whiles the KM was 500 ± 50 µM. NADH showed a markedly reduced rate of reduction with a kred of 0.34 ± 0.03 s⁻¹ with an apparent KM of 3000 ± 500 µM. The rate of flavin re-oxidation in the absence of monooxygenase IBAH was 4.79 × 10⁻⁹ M-1 s⁻¹. Our results suggest a reaction mechanism for the IBAH monooxygenase system controlled by the oxidation half reaction that may be modulated by a complex formation between the reductase and monooxygenase components. / Master of Science in Life Sciences