In vitro methanol production from methyl coenzyme M using the Methanosarcina barkeri MtaABC protein complex.

Methanol:coenzyme M methyltransferase is an enzyme complex composed of three subunits, MtaA, MtaB, and MtaC, found in methanogenic archaea and is needed for their growth on methanol ultimately producing methane. MtaABC catalyzes the energetically favorable methyl transfer from methanol to coenzyme M to form methyl coenzyme M. Here we demonstrate that this important reaction for possible production of methanol from the anaerobic oxidation of methane can be reversed in vitro. To this effect, we have expressed and purified the Methanosarcina barkeri MtaABC enzyme, and developed an in vitro functional assay that demonstrates MtaABC can catalyze the energetically unfavorable (ΔG° = 27 kJ/mol) reverse reaction starting from methyl coenzyme M and generating methanol as a product. Demonstration of an in vitro ability of MtaABC to produce methanol may ultimately enable the anaerobic oxidation of methane to produce methanol and from methanol alternative fuel or fuel-precursor molecules. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1243-1249, 2017.

Crystal Structure and Atomic Level Analysis of Plasma PAF-AH.

The structure of plasma PAF-AH was solved to a resolution of 1.5Å using X-ray crystallography. The enzyme has a classic α/ß serine hydrolase fold containing a catalytic triad of Ser273, Asp296, and His351. A hydrophobic patch of the enzyme involving two α-helices (114-126 and 362-369) and neighboring residues have been shown to be essential for lipoprotein particle binding by mutagenesis and mass spectrometry hydrogen/deuterium exchange experiments. An interface-bound model of the enzyme positions the active site above the hydrophobic-hydrophilic interface and is consistent with the known substrate specificity of the enzyme. Several ligand-bound structures of plasma PAF-AH have been solved with organophosphorus compounds and modeled with competitive inhibitors of high affinity and selectivity. This chapter presents an overview of the structure of plasma PAF-AH, molecular details of its functional role, and the interaction of the enzyme with lipoprotein particles.

Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A(2).

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) or PLA(2)G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA(2) has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA(2) have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA(2) inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA(2) and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA(2) in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA(2) function in biological systems.