122nd General Meeting of the KCS

Type Symposium
Area Bioinorganic Chemistry (Metals in Biology)
Room No. Room 323
Time THU 16:35-:
Code INOR1-3
Subject Regulation of hydroxylase activity through auxiliary components in metallo-enzymes
Authors Seung Jae Lee
Department of Chemistry and Institute for Molecular Biology and Genetics, Chonbuk National University, Korea
Abstract Hydroxylation through soluble methane monooxygenase (sMMO) has been studied extensively to understand the biological conversion from methane to methanol in ambient conditions, although more detailed mechanisms of this biocatalyst are yet undiscovered. In this study, sMMO components, including the hydroxylase (MMOH), regulatory (MMOB), and reductase (MMOR), were expressed and purified from a type II methanotroph, Methylosinus sporium strain 5 (M. sporium 5), to characterize its hydroxylation mechanism. Brown–black pigments were detected in M. sporium 5 during cell growth, with the optical density value of 8.0 at 600 nm (OD600), and this change was dependent on the ratio of cell:iron. The cell extract showed that MMOH, MMOB, and MMOR were expressed by M. sporium 5, and these enzymes were extracted with high purity. The enzyme activity confirmed that 2.0 mol equivalents of MMOB is necessary to achieve catalytic activities, and sMMO from M. sporium 5 oxidized a broad range of substrates, including alkanes, alkenes, halogens, and aromatics. Optimal activities were observed at pH 7.5 in most substrates, possibly because of the electron transfer environment in MMOR, and this was explained by the optical spectra from FAD-containing domains. The substitution of MMOB or MMOR from another type II methanotroph, Methylocystis species M (M. species M), retained specific enzyme activities, demonstrating the successful crossreactivity of M. sporium 5. These characterizations will provide fundamental information for further enzymatic studies to acquire biophysical information to elucidate sMMO mechanisms.
E-mail slee026@jbnu.ac.kr