Arginine methylation is a common posttranslational modification that is found on both histone and non-histone proteins. Three types of arginine methylation exist in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). PRMT1 is the primary methyltransferase that deposits the ADMA mark, and it accounts for over 90% of this type of methylation. Here, we show that with the loss of PRMT1 activity, there are major increases in global MMA and SDMA levels, as detected by type-specific antibodies. Amino acid analysis confirms that MMA and SDMA levels accumulate when ADMA levels are reduced. These findings reveal the dynamic interplay between different arginine methylation types in the cells, and that the pre-existence of the dominant ADMA mark can block the occurrence of SDMA and MMA marks on the same substrate. This study provides clear evidence of competition for different arginine methylation types on the same substrates.

Background: Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed transesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially. Results: We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the halfinactivation time at 50°C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis. Conclusions: Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties.

Background: Sortase enzymes catalyze a transpeptidation reaction that displays bacterial surface proteins. Results: Structural and computational studies reveal how the sortase B enzyme recognizes its sorting signal substrate. Conclusion: Sortase enzymes catalyze transpeptidation using a substrate-stabilized oxyanion hole. Significance: The results of this work could facilitate the rational design of sortase inhibitors.

Renewable lignocellulosic plant biomass is a promising feedstock from which to produce biofuels, chemicals, and materials. One approach to cost-effectively exploit this resource is to use consolidating bioprocessing (CBP) microbes that directly convert lignocellulose into valuable end products. Because many promising CBP-enabling microbes are non-cellulolytic, recent work has sought to engineer them to display multi-cellulase containing minicellulosomes that hydrolyze biomass more efficiently than isolated enzymes. In this review, we discuss progress in engineering the surfaces of the model microorganisms: Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. We compare the distinct approaches used to display cellulases and minicellulosomes, as well as their surface enzyme densities and cellulolytic activities. Thus far, minicellulosomes have only been grafted onto the surfaces of B. subtilis and S. cerevisiae, suggesting that the absence of an outer membrane in fungi and Gram-positive bacteria may make their surfaces better suited for displaying the elaborate multi-enzyme complexes needed to efficiently degrade lignocellulose.