Transcriptional regulation of soluble methane monooxygenase via enhancer-binding protein derived from Methylosinus sporium 5

Methane is a major greenhouse gas, and methanotrophs regulate the methane level in the carbon cycle. Soluble methane monooxygenase (sMMO) is expressed in various methanotroph genera, including Alphaproteobacteria and Gammaproteobacteria, and catalyzes the hydroxylation of methane to methanol. It has been proposed that MmoR regulates the expression of sMMO as an enhancer-binding protein under copper-limited conditions; however, details on this transcriptional regulation remain limited. Herein, we elucidate the transcriptional pathway of sMMO depending on copper ion concentration, which affects the interaction of MmoR and sigma factor. MmoR and sigma-54 (σ⁵⁴) from Methylosinus sporium 5 were successfully overexpressed in Escherichia coli and purified to investigate sMMO transcription in methanotrophs. The results indicated that σ⁵⁴ binds to a promoter positioned −24 (GG) and −12 (TGC) upstream between mmoG and mmoX1. The binding affinity and selectivity are lower (K_d = 184.6 ± 6.2 nM) than those of MmoR. MmoR interacts with the upstream activator sequence (UAS) with a strong binding affinity (K_d = 12.5 ± 0.5 nM). Mutational studies demonstrated that MmoR has high selectivity to its binding partner (ACA-xx-TGT). Titration assays have demonstrated that MmoR does not coordinate with copper ions directly; however, its binding affinity to UAS decreases in a low-copper-containing medium. MmoR strongly interacts with adenosine triphosphate (K_d = 62.8 ± 0.5 nM) to generate RNA polymerase complex. This study demonstrated that the binding events of both MmoR and σ⁵⁴ that regulate transcription in M. sporium 5 depend on the copper ion concentration.