Printable enzyme-embedded materials for methane to methanol conversion.

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scale structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas-liquid reactions.

Insights into the different dioxygen activation pathways of methane and toluene monooxygenase hydroxylases.

The methane and toluene monooxygenase hydroxylases (MMOH and TMOH, respectively) have almost identical active sites, yet the physical and chemical properties of their oxygenated intermediates, designated P*, H(peroxo), Q, and Q* in MMOH and ToMOH(peroxo) in a subclass of TMOH, ToMOH, are substantially different. We review and compare the structural differences in the vicinity of the active sites of these enzymes and discuss which changes could give rise to the different behavior of H(peroxo) and Q. In particular, analysis of multiple crystal structures reveals that T213 in MMOH and the analogous T201 in TMOH, located in the immediate vicinity of the active site, have different rotatory configurations. We study the rotational energy profiles of these threonine residues with the use of molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) computational methods and put forward a hypothesis according to which T213 and T201 play an important role in the formation of different types of peroxodiiron(III) species in MMOH and ToMOH. The hypothesis is indirectly supported by the QM/MM calculations of the peroxodiiron(III) models of ToMOH and the theoretically computed Mössbauer spectra. It also helps explain the formation of two distinct peroxodiiron(III) species in the T201S mutant of ToMOH. Additionally, a role for the ToMOD regulatory protein, which is essential for intermediate formation and protein functioning in the ToMO system, is advanced. We find that the low quadrupole splitting parameter in the Mössbauer spectrum observed for a ToMOH(peroxo) intermediate can be explained by protonation of the peroxo moiety, possibly stabilized by the T201 residue. Finally, similarities between the oxygen activation mechanisms of the monooxygenases and cytochrome P450 are discussed.

Diversity of soluble methane monooxygenase-containing methanotrophs isolated from polluted environments.

Methanotrophs were enriched and isolated from polluted environments in Canada and Germany. Enrichments in low copper media were designed to specifically encourage growth of soluble methane monooxygenase (sMMO) containing organisms. The 10 isolates were characterized physiologically and genetically with one type I and nine type II methanotrophs being identified. Three key genes: 16S rRNA; pmoA and mmoX, encoding for the particulate and soluble methane monooxygenases respectively, were cloned from the isolates and sequenced. Phylogenetic analysis of these sequences identified strains, which were closely related to Methylococcus capsulatus, Methylocystis sp., Methylosinus sporium and Methylosinus trichosporium. Diversity of sMMO-containing methanotrophs detected in this and previous studies was rather narrow, both genetically and physiologically, suggesting possible constraints on genetic diversity of sMMO due to essential conservation of enzyme function.

The NADH-dependent reductase of a putative multicomponent tetrahydrofuran mono-oxygenase contains a covalently bound FAD.

NADH-cytochrome c oxidoreductase activity specifically expressed during growth on tetrahydrofuran was detected in cell extracts of Pseudonocardia sp. strain K1. The enzyme catalyzing this reaction was purified to apparent homogeneity by a three-step purification procedure. It was characterized as a monomer of apparent molecular mass 40 kDa. Spectroscopic studies indicated that it contains an iron-sulfur cluster and a flavin cofactor. An amount of 1 mol of flavin and 1 mol of iron was determined per mol of homogeneous protein. The N-terminal amino-acid sequence exhibited great similarity to the reductase component of various oxygenases. Cloning and sequencing of the corresponding gene designated as thmD revealed an ORF encoding a protein of 360 amino acids. An overall similarity of up to 38% was obtained to the NAD(P)H-acceptor reductase of several binuclear iron-containing mono-oxygenases. Conserved sequence motifs were identified that were similar to the chloroplast-type ferredoxin 2Fe-2S centre and to nucleotide-binding domains. Studies on the flavin cofactor showed that it could not be removed from the protein by denaturation, indicating a covalent attachment. Spectroscopic studies revealed that the flavin is at the FAD level and covalently bound to the protein via the flavin 8alpha-methyl group. Thus, the isolated reductase component is the first enzyme of this type for which a covalent attachment of the flavin has been observed.