Molecular Dynamics Simulations of Enzymes with Quantum Mechanical/Molecular Mechanical Potentials

S-adenosyl methionine (SAM) dependent methylation process is universally found in all branches of life. It has important implications in mammalian pathogenesis and plant metabolism. The methyl transfer is normally catalyzed by SAM-dependent methyltransferases(MTases). Two MTases are studied in this dissertation: the 1,7-dimethylxanthine methyltransferase (DXMT) which involve in plant caffeine biosynthesis, and the protein arginine methyltransferase 5(PRMT5) that participates in eukaryotic posttranslational modification. The late phase of caffeine biosynthesis starts from the substrate xanthosine and ends with the product caffeine, with theobromine as an intermediate product. DXMT is a key enzyme in this process and catalyzes two methylation steps: 1)methylation of 7-methylxanthine to form theobromine; 2)methylation of theobromine to form caffeine. The catalytic mechanism and product promiscuity of DXMT is intriguing. In Chapter 1, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations were performed to explain the dual catalytic roles of DXMT. Simulation results show that a histidine residue may act as a general base catalyst during methylations. PRMTs can work as modifiers for histones and methylate the substrate arginine, thus interfering with histone code orchestration. The product specificity of PRMTs refers to their ability to produce either symmetric di-methylarginine(SDMA), asymmetric di-methylarginine(ADMA) or mono-methylarginine(MMA). Understanding the product specificity of PRMTs is important since different methylations may cause distinctive, even inverse biological consequences. PRMT5 produces SDMA, as compared to PRMT1 and PRMT3 that produce ADMA. In Chapter 2, simulations of PRMT5 have drawn a theoretical insight into the catalytic difference between SDMA and ADMA. Neddylation is a type of eukaryotic Ubiquitin-like (UBL) protein modification that is essential in cell division and development. Unlike ubiquitin and other small ubiquitin-like modifiers which target variety of protein substrates, the UBL NEDD8 is highly selective on modifying cullin proteins and contributes to 10% ~20% of all cellular ubiquitination and ubiquitination-like modification. In Chapter 3, the crystal structure of a trapped E3-E2 ̴ NEDD8-CUL1 intermediate was used for modeling, and simulations were applied to investigate the catalytic mechanism of NEDD8 transfer from E2 to the substrate. Some important insights were observed that may be used to understand the functional properties of the enzyme

Computer Simulations of Enzymes

Enzymes are important catalysts in living systems, and understanding catalytic mechanisms of enzymes is an important task for modern biophysics and biochemistry. Computer simulations have emerged as very useful tools for understanding how enzymes work. In this dissertation, QM/MM MD simulations were applied to study the catalytic mechanisms of several enzymes, including sedolisin, S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases, and salicylic acid binding protein 2. For sedolisin, we focus on the acylation and deacylation reactions catalyzed by the enzymes. We proposed a general acid/base mechanism involving the Glu/Asp residues at the active site. MD and QM/MM free energy simulations on pro-kumamolisin show that the protonation of Asp164 would be able to trigger conformational changes and generate the functional active site for autocatalysis. The free energy simulations reported for SAMT, an AdoMet-dependent methyltransferase, showed that while the structure of the reactant complex containing salicylate, its natural substrate, is rather close to the corresponding TS structure, this is not the case for 4-hydroxybenzoate. The simulations demonstrated that additional energy is required to generate the TS-like structure for 4-hydroxybenzoate, consistent with the low activity of the enzyme toward this substrate. For protein lysine methyltransferase SET7/9, we showed that while the wild type SET7/9 may act like a mono-methylase, the Y245→A mutation could increase the ability of SET7/9 to add two more methyl groups on the target lysine. The substrate specificity of salicylic acid binding protein 2 (SABP2) has also been studied during my graduate study. This enzyme has promiscuous esterase activity toward a series of substrates, but shows high activity toward its natural substrate methyl salicylate (MeSA). We demonstrated that SABP2 seems to represent a case in which the enzyme itself might have not been perfectly evolved and that substrate-assisted catalysis (SAC) involving its natural substrate may be used to enhance the activity and achieve substrate discrimination. In addition to enzymes, the prediction of protein-protein interactions (PPI) is also included in my dissertation. We established a robust pipeline for PPI prediction by integrating multiple classifiers using random forests algorithm. This pipeline could be very useful for predicting PPI

Multiscale modeling for complex chemical systems: Highlights about the Nobel Prize in Chemistry 2013

The Nobel Prize in Chemistry 2013 was awarded jointly to Martin Karplus, Michael Levitt and Arieh Warshel for the development of multiscale models for complex chemical systems. From the simplest approximation of molecular mechanics (MM) to quantum mechanics (QM), computational techniques allow simulating a great variety ofchemical systems. Combined QM/MM methodologies, however, are the best consensus for treating complex biological systems. Herein we review the theoretical basis of QM/MM methods and their applications during the last twenty years.

Functional Study of Plant SABATH Methyltransferases in the Biosynthesis of Methyl Cinnamate, Juvenile Hormone III and Methyl Gibberellins

SABATH family of methyltransferases (MTs) is a group of plant MTs that are capable of methylating phytohormones and other small molecular compounds. This dissertation investigates biochemical function and evolution of SABATH genes with special interests in the biosynthesis of methyl cinnamate, juvenile hormone III, and methyl gibberellins. Methyl cinnamate is a fragrant volatile compound that occurs in a variety of land plants including great scent liverwort, Conocephalum salebrosum Szweykowski, Buczkowska and Odrzykoski. Using a comparative transcriptomic approach, we compare biosynthesis in liverworts and flowering plants and identified a cinnamic acid methyltransferase (CAMT) from C. salebrosum SABATH MTs. Structural and phylogenetic evidence indicate that methyl cinnamate biosynthesis in liverwort and flowering plants originated through convergent evolution. Juvenile hormones (JHs) are important in insect development and reproduction, however, JH III has been detected in some plants including Cyperus iria L. Unlike JH III biosynthetic pathway in insects that has been largely resolved, our understanding of JH III biosynthesis in plants remains limited. To reveal key enzymes involved in JH III biosynthesis in plants, a comparative transcriptomic approach was undertaken and identified C. iriahomologs of those enzyme-coding genes in insects, but no homolog for the biosynthesis of methyl farnesoate, the immediate precursor of JH III in plants. However, C. iria SABATH MTs were used to identify a farnesoic acid methyltransferase (FAMT) that can produce methyl farnesoate.This discovery implied the independent evolution of JH III biosynthetic pathway in plants and insects. Gibberellins (GAs) are a class of plant hormones that have multiple roles in different physiological processes. GA methylation is a recently discovered route of maintaining GA homeostasis which is catalyzed by gibberellic acid methyltransferase (GAMT). Because GAMT genes have previously identified only in the model plant Arabidopsis, the importance of this GA catabolic mechanism in other plants has remained unclear. To investigate the distribution, evolution, and biochemical functions of GAMT genes in land plants, we systemically analyzed 260 plant genomes and found that the GAMT gene arose early in the evolution of seed plants and was subsequently got lost in many flowering plants