A cell wall synthase accelerates plasma membrane partitioning in mycobacteria.

Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced both by bilayer-intrinsic factors like molecular composition and by bilayer-extrinsic factors such as interactions with other membranes and solid supports. While cellular membranes can departition in response to bilayer-intrinsic or -extrinsic disruptions, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis spatially and biochemically departitions in response to the fluidizing agent benzyl alcohol, then repartitions upon fluidizer washout. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth during recovery from benzyl alcohol exposure. PonA2's role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. Active cell wall polymerization promotes de novo membrane partitioning and the completed cell wall polymer helps to maintain membrane partitioning. Our work highlights the complexity of membrane-cell wall interactions and establishes a facile model system for departitioning and repartitioning cellular membranes.

Electron withdrawing group-dependent substrate inhibition of an α-ketoamide reductase from Saccharomyces cerevisiae.

Aldo-keto reductases remain enzymes of interest in biocatalysis due to their ability to reduce carbonyls to alcohols stereospecifically. Based on genomic sequence, we identified aldo-keto reductases of a S. cerevisiae strain extracted from an ancient amber sample. One of the putative enzymes, AKR 163, displays 99% identity with α-amide ketoreductases from the S288C and YJM248 S. cerevisiae strains, which have been investigated for biocatalytic applications. To further investigate AKR 163, we successfully cloned, expressed in E.coli as a glutathione-S-transferase fusion protein, and affinity purified AKR 163. Kinetic studies revealed that AKR 163 experiences strong substrate inhibition by substrates containing halogen atoms or other electron withdrawing groups adjacent to the reactive carbonyl, with Ki values ranging from 0.29 to 0.6 mM and KM values ranging from 0.38 to 0.9 mM at pH 8.0. Substrates without electron withdrawing groups do not display substrate inhibition kinetics and possess much larger KM values between 83 and 260 mM under the same conditions. The kcat values ranged from 0.5 to 2.5s-1 for substrates exhibiting substrate inhibition and 0.22 to 0.52s-1 for substrates that do not engage in substrate inhibition. Overall, the results are consistent with rate-limiting dissociation of the NADP+ cofactor after hydride transfer when electron withdrawing groups are present and activating the reduction step. This process leads to a buildup of enzyme-NADP+ complex that is susceptible to binding and inhibition by a second substrate molecule.