Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that catalyses the conversion of methane to methanol. Knowledge of how pMMO performs this extremely challenging chemistry may have an impact on the use of methane as an alternative energy source by facilitating the development of new synthetic catalysts. We have determined the structure of pMMO from the methanotroph Methylococcus capsulatus (Bath) to a resolution of 2.8 A. The enzyme is a trimer with an alpha3beta3gamma3 polypeptide arrangement. Two metal centres, modelled as mononuclear copper and dinuclear copper, are located in soluble regions of each pmoB subunit, which resembles cytochrome c oxidase subunit II. A third metal centre, occupied by zinc in the crystal, is located within the membrane. The structure provides new insight into the molecular details of biological methane oxidation.

SummaryUpon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.

Gaucher disease results from mutations in the lysosomal enzyme acid beta-glucosidase (GCase). Although enzyme replacement therapy has improved the health of some affected individuals, such as those with the prevalent N370S mutation, oral treatment with pharmacological chaperones may be therapeutic in a wider range of tissue compartments by restoring sufficient activity of endogenous mutant GCase. Here we demonstrate that isofagomine (IFG, 1) binds to the GCase active site, and both increases GCase activity in cell lysates and restores lysosomal trafficking in cells containing N370S mutant GCase. We also compare the crystal structures of IFG-bound GCase at low pH with those of glycerol-bound GCase at low pH and apo-GCase at neutral pH. Our data indicate that IFG induces active GCase, which is secured by interactions with Asn370. The design of small molecules that stabilize substrate-bound conformations of mutant proteins may be a general therapeutic strategy for diseases caused by protein misfolding and mistrafficking.

Particulate methane monooxygenase (pMMO) is a membranebound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria. Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance. Difficulties in solubilizing and purifying active pMMO have led to conflicting reports regarding its biochemical and biophysical properties, however. We have purified pMMO from Methylococcus capsulatus (Bath) and detected activity. The purified enzyme has a molecular mass of Ϸ200 kDa, probably corresponding to an ␣2␤2␥2 polypeptide arrangement. Each 200-kDa pMMO complex contains 4.8 ؎ 0.8 copper ions and 1.5 ؎ 0.7 iron ions. Electron paramagnetic resonance spectroscopic parameters corresponding to 40 -60% of the total copper are consistent with the presence of a mononuclear type 2 copper site. X-ray absorption near edge spectra indicate that purified pMMO is a mixture of Cu(I) and Cu(II) oxidation states. Finally, extended x-ray absorption fine structure data are best fit with oxygen͞nitrogen ligands and a 2.57-Å Cu-Cu interaction, providing direct evidence for a copper-containing cluster in pMMO. O ne of the great challenges for the chemical and engineering communities is the selective oxidation of methane to methanol. Although world reserves of petroleum and natural gas are comparable, methane is used far less efficiently as an energy source because it has a low energy density and is hazardous and expensive to transport (1). Conversion of methane to a liquid such as methanol would solve this problem, but current industrial processes for this transformation are costly and inefficient, requiring high temperatures and pressures (2, 3). By contrast, methanotrophic bacteria (4) oxidize methane to methanol at ambient temperature and pressure by using methane monooxygenase (MMO) enzyme systems (5). Only one other enzyme, ammonia monooxygenase (6), can activate the COH bond in methane (104 kcal͞mol). Cytochromes P450 (7) and copper monooxygenases (8) oxidize larger, more reactive hydrocarbons, but cannot hydroxylate methane. Therefore, a detailed understanding of biological methane oxidation is of both fundamental chemical and potential commercial importance.All methanotrophs produce a membrane-bound MMO called particulate MMO (pMMO) (5). Under conditions of low copper availability, several strains also express a soluble enzyme (sMMO) (9), which contains a catalytic diiron center (10). Whereas the structure, biochemistry, and mechanism of sMMO are well understood (11), studies of pMMO are less advanced because of difficulties in solubilizing and purifying active enzyme. The Methylococcus capsulatus (Bath) pMMO comprises three polypeptides, the ␣ (Ϸ47 kDa), ␤ (Ϸ24 kDa), and ␥ (Ϸ22 kDa) subunits, encoded by the pmoB, pmoA, and pmoC genes, respectively (12, 13). It is not known how these three polypeptides are arranged in the pMMO holoenzyme. According to radiolabeling experiments with the suicide substrate acetylene, the active ...