|Subject||alphaproteobacteria; bacteria; biotechnology; cloning, molecular; copper; DNA; DNA, bacterial; enzyme stability; enzymology; genes; genes, bacterial; genetics; growth & development; hydrocarbons; isolation & purification; metabolism; methane; methods; methylococcus; nitrogen; oxygenases; phylogeny; polymerase chain reaction; protein; ribosomal; RNA; RNA, ribosomal, 16S; sequence analysis, DNA; solubility; substrate specificity|
Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the ﬁrst step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was puriﬁed to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria conﬁrmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 μM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate speciﬁcity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an α₂β₂γ₂ structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is stronglyinhibited by Hg²⁺ ions (with a total loss of enzyme activity at 0.01 mM Hg²⁺) and Cu²⁺, Zn²⁺, and Ni²⁺ ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).