Aims: Particulate methane monooxygenase (pMMO) is an integral membrane protein that converts methane to methanol as the first step in the metabolic pathway of methanotroph bacteria. Methanotroph have a slow growth rate that make researcher have to develop an alternative approach by expressing the pMMO genes in Escherichia coli. However, it was very difficult to express all the pMMO encoded genes in E. coli and it is suspected that the protein might be toxic to E. coli. Therefore, this research tried another approach by expressing the active site of pMMO enzyme; cupredoxin domain of pmoB subunit encoded by spmoB gene. Methodology and results: The spmoB gene from Methylococcus capsulatus (Bath) was expressed in E. coli BL21 (DE3) under T7 promoter and pET15b as the expression vector. Several modifications were made so this gene would be expressed in the cytoplasm. Expression analysis with SDS-PAGE showed that overexpression of this gene could be done at several concentrations of IPTG and incubation temperature. The spmoB gene expression produced a recombinant protein with a size approximately 38.9 kDa. Assay of spmoB protein activity showed that the amount of methanol accumulated during methane oxidation by the recombinant strain was 0.114 mmol/mL culture.h. Conclusion, significance and impact study: We successfully expressed spmoB gene in E. coli BL21 (DE3) without high production of toxic compounds and it has methane oxidation activity. This result allowed further characterization of its potential applications.

Ubiquitination is a key signal for endocytosis of proteins on the plasma membrane. The ubiquitin ligase Rsp5 of Saccharomyces cerevisiae, which contains an amino-terminal membrane-binding C2 domain, three substrate-recognizing tryptophan-tryptophan (WW) domains and a carboxyl-terminal catalytic homologous to the E6-AP carboxyl terminus (HECT) domain, can ubiquitinate plasma membrane proteins directing them for endocytosis. Here, we examined the roles of the C2 domain in endocytosis for the downregulation of the general amino acid permease Gap1, which is one of nitrogen-regulated permeases in S. cerevisiae. First, we constructed several rsp5 mutants producing Rsp5 variants without the C2 domain or with amino acid changes of membrane-binding lysine residues. These mutants showed defects in endocytosis of Gap1 in response to a preferred nitrogen source. Intriguingly, we found that ubiquitination of Gap1 in these mutant cells was highly similar to that in wild-type cells during endocytosis. These results indicate that the C2 domain is essential for endocytosis but not for ubiquitination of substrates such as Gap1. Moreover, genetic and biochemical analyses showed that the endocytic protein Rvs167 was ubiquitinated via Rsp5 and the C2 domain was required for efficient ubiquitination in response to a preferred nitrogen source. Here, we propose a mechanism for the C2 domain-mediated endocytosis of plasma membrane permeases.