Probing the Hydrophobic Pocket of the Active Site in the Particulate Methane Monooxygenase (pMMO) from Methylococcus capsulatus (Bath) by Variable Stereoselective Alkane Hydroxylation and Olefin Epoxidation

Abstract

pMMO from M. capsulatus (Bath) oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides. According to experiments performed with cryptically chiral ethane and D,L-[2-(2)H(1),3-(2)H(1)]butane, the reactions proceed through the concerted O-atom insertion mechanism. However, when propene and but-1-ene are used as epoxidation substrates, the enantiomeric excesses (ees) of the enzymatic products are only 18 and 37 %, respectively. This relatively poor stereoselectivity in the enzymatic epoxidation presumably reflects low stereochemical differentiation between the re and si faces in the hydrophobic pocket of the active site. Further insights into the reaction mechanism are now provided by studies on trans-but-2-ene, which reveal only the D,L-2,3-dimethyloxirane products, and on cis-but-2-ene, which yield only the meso product. These observations indicate that the enzymatic epoxidation indeed proceeds through electrophilic syn addition. To achieve better facial selectivity, we have also used 3,3,3-trifluoroprop-1-ene as the substrate. The products obtained are 90 % (2S)-oxirane. When 1,1,1-trifluoropropane is the substrate, the hydroxylation at the 2-carbon exhibits an inverse chiral selectivity relative to that seen with normal butane, if we consider the size of the CF(3) group in the fluorinated propane to be comparable to one of the ethyl groups in butane. These experiments are beginning to delineate the factors that influence the orientations of various substrates in the hydrophobic cavity of the active site in the enzyme.

Inhibitory Effects of C 2 to C 10 1-Alkynes on Ammonia Oxidation in Two Nitrososphaera Species

“…It has been known for many years that pMMOs of the well-studied methanotrophs, Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), exhibit a hydrocarbon substrate range that extends beyond CH 4 and yet, are restricted to short-chain alkanes (C 2 to C 5 ) and alkenes (C 2 to C 4 ) (27)(28)(29). It has been suggested that the chain-length-limited substrate range of pMMO is due to the active site of the enzyme being located in a hydrophobic pocket that cannot accommodate alkanes of ϾC 5 , and there is speculation that the pocket might have separate binding sites for CH 4 versus the larger substrates (28)(29)(30)(31). Perhaps, it is not unreasonable to link our own data with the above-mentioned properties of pMMO to suggest that AOA AMO might also contain a binding pocket resembling pMMO that is either too small to freely accommodate ՆC 6 1-alkynes or constrained sufficiently to allow binding but does not allow turnover of the longer 1-alkynes.…”

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene

“…Thus, although the X-ray crystal structures of pMMO do not contain the full complement of metal cofactors as in the active enzyme embedded within the lipid bilayer, the availability of these structures can still offer the opportunity to identify potential substrate-binding sites in the transmembrane domain by computer docking experiments [34,35]. However, it is important to point out here that while the docking experiments are sufficiently accurate to locate the binding sites of small molecules in the protein fold as determined by the X-ray structure, the results pertain only to the "static" fold of the enzyme in its "native" structure.…”

“…In addition, they all dock into the cavity close to the putative tricopper site (site D) of the enzyme [34]. Thus, it would seem that the pMMO uses the same substrate pathway(s) to deliver hydrocarbon substrates including the suicide substrate HCCH to the active site of the enzyme.…”

A Physical Chemist's Expedition to Explore the World of Membrane Proteins

“…In our work, a preparation of the enzyme with the full complement of 15 Cu ions was always used (26). At the active site of this enzyme is a trinuclear copper cluster (24,26,68) that, upon activation by molecular oxygen, mediates the transfer of a singlet oxene to the methane at the active site. A recent crystal structure of pMMO from M. capsulatus did not reveal a trinuclear copper center in the structure (60), but the protein preparation on which the crystal structure was based did not possess enzyme activity.…”

“…Those of us who conduct biophysical measurements are often less circumspect about the quality of the sample than they should be. Fortunately, in the case of pMMO, it was possible to reconcile the biochemical/biophysical data with the crystal structure by introducing the missing metallic cofactors back into the protein scaffold (24,26,68) (Figure 3).…”