Recovery of particulate methane monooxygenase structure and activity in a lipid bilayer

Abstract

Bacterial methane oxidation using the enzyme particulate methane monooxygenase (pMMO) contributes to the removal of environmental methane, a potent greenhouse gas. Crystal structures determined using inactive, detergent-solubilized pMMO lack several conserved regions neighboring the proposed active site. We show that reconstituting pMMO in nanodiscs with lipids extracted from the native organism restores methane oxidation activity. Multiple nanodisc-embedded pMMO structures determined by cryo–electron microscopy to 2.14- to 2.46-angstrom resolution reveal the structure of pMMO in a lipid environment. The resulting model includes stabilizing lipids, regions of the PmoA and PmoC subunits not observed in prior structures, and a previously undetected copper-binding site in the PmoC subunit with an adjacent hydrophobic cavity. These structures provide a revised framework for understanding and engineering pMMO function.

Distance-based global analysis of consistentcis-bonds in protein backbones

“…This protein does not have a UniProt ID but has an NCBI accession of WP_036287217. As a recent cryo-electron microscopy structure (PDB ID: 7S4M) of this subunit only contains six cis bonds (Koo et al, 2022), the consistency of these 10 cis bonds needs to be confirmed. The second largest number (nine) of consistent cis bonds is found in GH43 arabinofuranosidase (AXHd3) of 529 amino acids (97.6% of the full length) from Humicola insolens , which also does not have a UniProt ID.…”

A Copper Cage‐Complex as Mimic of the pMMO CuC Site

“…For copper, a resolution of 2.16 Å has been obtained and for pMMO with a zinc occupied Cu C site a structure with 2.80 Å resolution has been reported. [8,19] For the structures of the enzymes, metal-nitrogen bond lengths within a range between 2.0 and 2.1 Å have been observed. We observe CuÀ N bond lengths of 1.8 Å and 2.1 Å in 1-Cu and 1.9 Å in [1-Cu]PF 6 •2 (THF).…”

“…[6][7][8] Copper occupancy of PmoC was shown to be crucial for catalytic activity in 2019 already, [9] and very recently again by cryo-electron microscopy studies on samples that still showed catalytic activity. [8] The activity goes along with an unusually high preservation of the enzyme structure, which also enables observation of the PmoC region by imaging techniques, and that for the first time in the absence of zinc. [10] Thereby, the already widely accepted constellation of Cu C with coordination of two imidazole and one carboxylate was confirmed (see Figure 1).…”

Tailoring the squalene-hopene cyclase for stereoconvergent and efficient cationic cyclization cascades

“…This inseparable relationship was recently demonstrated in the reconstitution of the in vitro activity of the membrane-bound particulate monooxygenase by mimicking the natural lipid environment. [68] Due to their inherent hydrophobicity, terpenes diffuse into the cell membrane presumably causing local perturbations, whereupon the enzyme reacts downregulating its activity. This regulatory function by sensing membrane integrity would be a simple tool for nature to control activity of membrane-bound enzymes as already hypothesized in 1975.…”