| Enzymes are the foundation of all life activities. All cells in a living organism are distinctive biochemical systems, in which thousands of biochemical reactions will take place at all times. When we eat, drink, breath or sleep, our body is always involved in countless biochemical reactions governed by enzymes. These enzymes are best catalysts in natural systems, which outperform almost synthetic catalysts. Compared with the general catalysts, the enzyme can perform highly efficient catalysis at the physiological temperature and pressure. Enzymes are the best teachers for chemists. Through the studies of structure and function of enzymes, scientists can deeply explore the enzymatic properties, which can further expand the applications of enzymes in chemical industry and our daily life. One the problem in research field of enzymatic reaction is that the chemical reaction is so fast that many details of reaction mechanism are hard to be captured by experimental means. In recent years, with the development of computer technology and the maturing of theoretical chemistry methods, we can perform accurate theoretical calculations on large biochemical systems.In this dissertation, we employed the combined quantum mechanics/molecular mechanics (QM/MM) method to systematically explore the catalytic mechanisms of several important iron-containing enzymes and sugar-related enzymes. According to the QM/MM calculations, we analyzed the interaction between the enzyme and substrate, captured the details of enzymatic reaction, and identified the important role some active site residues. These results not only are in well agreement with some experimental results, but further supplement the experiment. Moreover, the calculated results can provide many useful insights into studies of catalytic mechanism and practical application of other related enzymes.The main research works of this dissertation are as followed:(1) Theoretical insights into catalytic mechanism of carbapenem synthase (CarC).Carbapenems, one subclass of β-lactam antibiotics, have been widely used in human medicine in the past decade. They retain a broad spectrum of antibacterial activity and remarkable potency against both Gram-positive and Gram-negative bacteria. However, recent studies clearly showed that the bacterial resistance to carbapenems is increasing. A complete understanding of the biosynthesis of carbapenem, thus, is critically important for designing new drug variants to combat resistance. Earlier biochemical studies found that the simplest natural carbapenem, carbapenem-3-carboxylic acid, is biosynthesized by the actions of three distinct enzymes in the phylogenetically distant plant pathogen Pectobacterium carotovorum. One of them is carbapenem synthase (CarC), which catalyzes the C5-epimerization and C2/3-desaturation of (3S,5S)-carbapenam to produce (5R)-carbapenem. Based on the first crystal structure of CarC reported by Clifton group, both experiments and theoretical calculations proposed the possible epimerization and desaturation mechanisms. However, given the imperfection of this structure (two loop segments surrounding the active site were missed), the results of these studies appear to be suspect. Recently, the complete crystal structure of CarC was reported, allowing us to perform accurate QM/MM calculations to explore the detailed reaction mechanism. We first analyzed the dioxygen binding site on metal and identified that the FeⅣ-oxo species has two potential orientations with either the oxo group trans to His101 or trans to His251. The former is energetically unstable, which can rapidly isomerize into the latter by rotation of the oxo group. Arg279 plays important roles in regulating the dioxygen binding and assisting the isomerization of FeⅣ-oxo species. The calculation results clearly support the stepwise C5-epimerization and C2/3-desaturation processes, involving two complete oxidative cycles. The epimerization process converts (3S,55)-carbapenam to the initial product (3S,5S)-carbapenam, undergoing H5 atom abstraction, inversion of C5-radical and reconstitution of the inverted C5-H bond. In the desaturation process, (3S,5R)-carbapenam rebinds the CarC active site with a new orientation. The desaturation across C2-C3 occurs without involving any active site residue other than the FeⅣ=O center. (3S,5R)-carbapenam is a substrate superior to its epimer for CarC to produce (5R)-carbapenem by efficient desaturation. Besides, the substrate hydro xylations compete with the target epimerization and desaturation reactions.(2) Theoretical insights into catalytic mechanism of engineered P450BM3 enzyme.Cytochrome P450 naturally catalyzed the P2-mediated C/N-H activation, epoxidation of alkenes, and oleefination of aldehyde/ketone. Similarity with enzymatic epoxidation of alkenes, organic syntheses usually obtain the cyclopropanation products by transferring the isoelectronic carbene to alkenes, which is almost impossible to occur in vivo. Inspired by region- and stereoselectivity of P450 enzymes, Arnold group first reported that the engineered P450BM3 variants have the ability to catalyze the cyclopropanation of alkenes with highly diastereo-and enantioselectivity. Except for the cyclopropanation, recent studies further found that the engineered P450 enzymes can catalyze the C-H amination, imidation of sulfides, and intermolecular aziridination. These studies not only enrich the applications of p450, but provide a solid basis for the experimental design of other new enzymes that catalyze the unusual reactions in natural system. Whereas the experimental studies have established the feasibility of cyclopropanation by designing some P450 variants and using different diazoester reagents and styrenyl substrates, there is no deep exploration for the cyclopropanation mechanism and nature of stereoselectivity. We utilized the QM-only and QM/MM calculations to investigate the engineered P450BM3-catalyzed cyclopropanation mechanism and stereoselectivity. The QM calculations suggested that no matter the axial ligand for Fe-porphyrin is thiol or hydroxyl group, the ground state of Fe-porphyrin carbene is triplet spin state, which is in agreement with QM/MM calculations. On the triplet state potential energy surface, Fe-porphyrin carbene intermediate is highly reactive to react with styrene to yield a C-radical intermediate. The C-radical subsequently attack the Fe-C bond to form cyclopropanation products. The stereoselectivity originate from four different relative orientations of diazoester reagents and styrene in enzyme active site, which is not possible to be achieved by rotating the phenyl or ester group from one specific orientation during the reaction. The calculated results further suggested that the axial cysteine-to-serine substitution in P450BM3-CIS will enhances the cis selectivity, which is in well agreement with experiments. These results uncover the nature of cyclopropanation stereoselectivity, which can provide beneficial guidance for the experimental redesign of P450BM3 variants to improve the yield and stereoselectivity.(3) Theoretical insights into catalytic mechanism of UDP-linked sugar N-acetyltransferase (WlbB).N-Acetyltransferases (NATs), one of the largest enzyme superfamilies, catalyze the transfer of the acetyl group fromacetyl coenzyme A (acetyl-CoA) to the amine group of various acceptor substrates ranging from simple small molecules to large proteins. NATs have been shown to be indispensable to numerous physiological processes, such as the activation and detoxification of carcinogens in humans, the inactivation of amino glycoside antibiotics in bacteria, the post-translational modification of histone proteins and the circadian rhythm in vertebrates. ManNAc3NAcA, a reasonably rare di-N-acetylated deoxysugar, is found in the outer membranes of some Gram-negative pathogenic bacteria. The precursor of ManNAc3NAcA is UDP-ManNAc3NAcA, which is biosynthesized by the actions of five distinct enzymes, starting from UDP-GlcNAc. The fourth enzyme, also referred to as WIbB in Bordetella petrii, is an N-acetyltransferase that catalyzes the biotransformation of UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NA) into UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NAcA). Based on the crystal structure, the detailed reaction mechanism of WlbB has been studied by QM/MM method. The QM/MM calculations propose a substrate-assisted acetylation mechanism, in which no active site residue is involved. Acetylation reaction undergoes a negatively charged CoA and a positively charged intermediate, rather than an oxyanion tetrahedral intermediate proposed by experiments. Whereas Asn84 is not essential for catalysis, it is not a residue to be trifled with. Moreover, the cautious selection of initial geometries was found to be important for exploring the enzymatic mechanism and getting reliable energy barriers of the reaction pathways.(4) Theoretical insights into catalytic mechanism of dTDP-glucose 4,6-dehydratase (DesⅣ).Deoxy sugars are an important class of carbohydrates that are frequently found in glycoproteins, lipopolysaccharides and glycolipids of bacterial cell surfaces and many secondary metabolites. D-desosamine is common deoxy sugar, which not only are indispensable to numerous biochemical processes, but is the important component of some macrolide antibiotics, such as neomethymycin, methymycin, picromycin, and erythromycin. Removal of the deoxy sugars from these antibiotics often decreases or even abolishes their pharmacological properties. The dTDP-glucose 4,6-dehydratase, also referred to as DesIV in Streptomyces venezuelae, catalyze the second step in the dTDP-D-desosamine biosynthetic pathway, which converts of dTDP-glucose into dTDP-4-keto-6-deoxy-glucose. Although many strides have been made in understanding the dTDP-glucose 4,6-dehydratase, mechanistic studies are still controversial. We have utilized the QM/MM approach to investigate the detailed mechanism of DesIV. The QM/MM calculations revise the previously proposed three-step sequential mechanism, which contains four enzymatic elementary reactions and one non-enzymatic enol-keto tautomerization reaction. The oxidation step proceeds through a concerted asynchronous mechanism with a calculated free energy barrier of 21.1 kcal/mol. The dehydration step prefers to a stepwise mechanism rather than a concerted mechanism, which involves an enolate intermediate. Two highly conserved residues Glul29 and Asp128 are involved in this step. In the reduction step, NADH returns the hydride back to glycosyl C6 and the phenolic hydroxyl of Tyr151 spontaneously denotes its proton to the C4-keto group, forming an enol sugar as the enzymatic product. After dissociated from the dehydratase active site and diffused into the solution, this enol sugar will facilely rearrange to give the more favorable dTDP-4-keto-6-dexoyglucose product. These calculation results may provide new inspiration for the mechanistic studies of other dTDP-glucose 4,6-dehydratases.(5) Theoretical insights into catalytic mechanism of UDP-GlcNAc 5,6-dehydratase (TunA).UDP-GlcNAc 5,6-dehydratase (TunA), a particular member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the bioconversion of UDP-GlcNAc to UDP-6-deoxy-GlcNAc-5,6-ene. TunA is one of the essential enzymes in the biosynthesis pathway of tunicamycns in S. chartreusis. Tunicamycns are important fatty acyl nucleoside antibiotics, which are well-established inhibitors of eukaryotic protein N-linked glycosylation. Moreover, tunicamycins can reversibly inhibit bacterial and mammalian phosphosugar transferases and potently block the N-palmitoylation of acyl-proteins. To elucidate the detailed mechanism of TunA, combined QM/MM calculations have been performed. Similarity with DesIV, the TunA-catalyzed process can also be divided into oxidation, dehydration and reduction steps. The oxidation step undergoes a concerted but asynchronous mechanism with an energy barrier of 21.6 kcal/mol. The dehydration step proceeds via the E1cB mechanism, involving proton transfer and departure of hydroxyl group. Glul21 firstly serves as the catalytic base, and then as acid to take part in the elimination of water. Whereas Cys120 is not directly involved in the dehydration reaction, it is confirmed to play an important role in maintaining the active pocket arrangement. As the reverse process of the oxidation reaction, the reduction step follows the similar concerted asynchronous mechanism, but the hydride transfer is prior to proton transfer.(6) Theoretical insights into catalytic mechanism of pectate lyase (BsPel).Pectin is the essential component of plant cell wall and is involved in various biochemical processes. This polymer is also widely used as thickening agent or stabilizer in the food industry. The degradation of pectin by bacteria requires the combination of several distinct enzymes. Pectate lyase is one of the indispensable enzymes to depolymerize pectin during soft rotting, which catalyze the random cleavage of α-1,4 glycosidic linkage of polygalacturonate via a β-elimination mechanism to generate the 4,5-desaturated oligogalacturonates. We report here an extensive QM/MM study on the detailed anti-β-elimination mechanism of Bacillus subtilis pectate lyase (BsPel). Residue Arg279 acts as the catalytic base to abstract α-proton from substrate, forming an energetically unstable carbanion intermediate. The C4-O4 bond of this intermediate is subsequently cleaved to generate a 4,5-unsaturated product and a negatively charged β-leaving group. Active site Ca2+ ions can efficiently lower the pKa value of a-proton, which facilitates the proton transfer. The proton source of β-leaving group is neither the Arg284 nor other active site residues, but most likely is the solvent water molecule. The calculated results further suggest that the careful selections of QM and Active region are crucial for exploring the enzymatic mechanism. |
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