Rubisco Adaptation Is More Limited by Phylogenetic Constraint Than by Catalytic Trade-off.

Rubisco assimilates CO2 to form the sugars that fuel life on earth. Correlations between rubisco kinetic traits across species have led to the proposition that rubisco adaptation is highly constrained by catalytic trade-offs. However, these analyses did not consider the phylogenetic context of the enzymes that were analyzed. Thus, it is possible that the correlations observed were an artefact of the presence of phylogenetic signal in rubisco kinetics and the phylogenetic relationship between the species that were sampled. Here, we conducted a phylogenetically resolved analysis of rubisco kinetics and show that there is a significant phylogenetic signal in rubisco kinetic traits. We re-evaluated the extent of catalytic trade-offs accounting for this phylogenetic signal and found that all were attenuated. Following phylogenetic correction, the largest catalytic trade-offs were observed between the Michaelis constant for CO2 and carboxylase turnover (∼21-37%), and between the Michaelis constants for CO2 and O2 (∼9-19%), respectively. All other catalytic trade-offs were substantially attenuated such that they were marginal (<9%) or non-significant. This phylogenetically resolved analysis of rubisco kinetic evolution also identified kinetic changes that occur concomitant with the evolution of C4 photosynthesis. Finally, we show that phylogenetic constraints have played a larger role than catalytic trade-offs in limiting the evolution of rubisco kinetics. Thus, although there is strong evidence for some catalytic trade-offs, rubisco adaptation has been more limited by phylogenetic constraint than by the combined action of all catalytic trade-offs.

Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5.

Protein arginine methyltransferases (PRMTs) are of great interest for the development of therapeutics due to their involvement in a number of malignancies, such as lung and colon cancer. PRMT5 catalyzes the formation of symmetrical dimethylarginine of a wide variety of substrates and is responsible for the majority of this mark within cells. To gain insight into the mechanism of PRMT5 inhibition, we co-expressed the human PRMT5:MEP50 complex (hPRMT5:MEP50) in insect cells for a detailed mechanistic study. In this report, we carry out steady state, product, and dead-end inhibitor studies that show hPRMT5:MEP50 uses a rapid equilibrium random order mechanism with EAP and EBQ dead-end complexes. We also provide evidence of ternary complex formation in solution using hydrogen/deuterium exchange mass spectrometry. Isotope exchange and intact protein mass spectrometry further rule out ping-pong as a potential enzyme mechanism, and finally, we show that PRMT5 exhibits a pre-steady state burst that corresponds to an initial slow turnover with all four active sites of the hetero-octamer being catalytically active.

Structural and Molecular Insight into Resistance Mechanisms of First Generation cMET Inhibitors.

Many small molecule inhibitors of the cMET receptor tyrosine kinase have been evaluated in clinical trials for the treatment of cancer and resistance-conferring mutations of cMET are beginning to be reported for a number of such compounds. There is now a need to understand specific cMET mutations at the molecular level, particularly concerning small molecule recognition. Toward this end, we report here the first crystal structures of the recent clinically observed resistance-conferring D1228V cMET mutant in complex with small molecule inhibitors, along with a crystal structure of wild-type cMET bound by the clinical compound savolitinib and supporting cellular, biochemical, and biophysical data. Our findings indicate that the D1228V alteration induces conformational changes in the kinase, which could have implications for small molecule inhibitor design. The data we report here increases our molecular understanding of the D1228V cMET mutation and provides insight for future inhibitor design.