Hansel Gómez

Retaining Glycosyltransferase Mechanism Studied by QM/MM Methods: Lipopolysaccharyl-α-1,4-galactosyltransferase C Transfers α-Galactose via an Oxocarbenium Ion-like Transition State

Glycosyltransferases (GTs) catalyze the highly specific biosynthesis of glycosidic bonds and, as such, are important both as drug targets and for biotechnological purposes. Despite their broad interest, fundamental questions about their reaction mechanism remain to be answered, especially for those GTs that transfer the sugar with net retention of the configuration at the anomeric carbon (retaining glycosyltransferases, ret-GTs). In the present work, we focus on the reaction catalyzed by lipopolysaccharyl-α-1,4-galactosyltransferase C (LgtC) from Neisseria meningitides. We study and compare the different proposed mechanisms (S(N)i, S(N)i-like, and double displacement mechanism via a covalent glycosyl-enzyme intermediate, CGE) by using density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations on the full enzyme. We characterize a dissociative single-displacement (S(N)i) mechanism consistent with the experimental data, in which the acceptor substrate attacks on the side of the UDP leaving group that acts as a catalytic base. We identify several key interactions that help this front-side attack by stabilizing the transition state. Among them, Gln189, the putative nucleophile in a double displacement mechanism, is shown to favor the charge development at the anomeric center by about 2 kcal/mol, compatible with experimental mutagenesis data. We predict that using 3-deoxylactose as acceptor would result in a reduction of k(cat) to 0.6-3% of that for the unmodified substrates. The reactions of the Q189A and Q189E mutants have also been investigated. For Q189E, there is a change in mechanism since a CGE can be formed which, however, is not able to evolve to products. The current findings are discussed in the light of the available experimental data and compared with those for other ret-GTs.

Enzyme-Directed Mutasynthesis: A Combined Experimental and Theoretical Approach to Substrate Recognition of a Polyketide Synthase

Acyltransferase domains control the extender unit recognition in Polyketide Synthases (PKS) and thereby the side-chain diversity of the resulting natural products. The enzyme engineering strategy presented here allows the alteration of the acyltransferase substrate profile to enable an engineered biosynthesis of natural product derivatives through the incorporation of a synthetic malonic acid thioester. Experimental sequence-function correlations combined with computational modeling revealed the origins of substrate recognition in these PKS domains and enabled a targeted mutagenesis. We show how a single point mutation was able to direct the incorporation of a malonic acid building block with a non-native functional group into erythromycin. This approach, introduced here as enzyme-directed mutasynthesis, opens a new field of possibilities beyond the state of the art for the combination of organic chemistry and biosynthesis toward natural product analogues.

Multiscale simulation of DNA

DNA is not only among the most important molecules in life, but a meeting point for biology, physics and chemistry, being studied by numerous techniques. Theoretical methods can help in gaining a detailed understanding of DNA structure and function, but their practical use is hampered by the multiscale nature of this molecule. In this regard, the study of DNA covers a broad range of different topics, from sub-Angstrom details of the electronic distributions of nucleobases, to the mechanical properties of millimeter-long chromatin fibers. Some of the biological processes involving DNA occur in femtoseconds, while others require years. In this review, we describe the most recent theoretical methods that have been considered to study DNA, from the electron to the chromosome, enriching our knowledge on this fascinating molecule.

Ligand induced formation of transient dimers of mammalian 12/15-lipoxygenase: A key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15‐LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer–dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding‐site model, we proposed that the binding of allosteric effectors may shift the monomer–dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)‐hydroxyoctadeca‐9(Z),11(E)‐dienoic acid] on the structural properties of rabbit 12/15‐LOX by small‐angle X‐ray scattering. Our data indicate that the enzyme undergoes ligand‐induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15‐LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild‐type LOX, and this structural alteration may lead to functional distortion of mutant enzymes. Proteins 2011.

A Native Ternary Complex Trapped in a Crystal Reveals the Catalytic Mechanism of a Retaining Glycosyltransferase

Glycosyltransferases (GTs) comprise a prominent family of enzymes that play critical roles in a variety of cellular processes, including cell signaling, cell development, and host-pathogen interactions. Glycosyl transfer can proceed with either inversion or retention of the anomeric configuration with respect to the reaction substrates and products. The elucidation of the catalytic mechanism of retaining GTs remains a major challenge. A native ternary complex of a GT in a productive mode for catalysis is reported, that of the retaining glucosyl-3-phosphoglycerate synthase GpgS from M. tuberculosis in the presence of the sugar donor UDP-Glc, the acceptor substrate phosphoglycerate, and the divalent cation cofactor. Through a combination of structural, chemical, enzymatic, molecular dynamics, and quantum-mechanics/molecular-mechanics (QM/MM) calculations, the catalytic mechanism was unraveled, thereby providing a strong experimental support for a front-side substrate-assisted SN i-type reaction.

Compaction of Duplex Nucleic Acids upon Native Electrospray Mass Spectrometry

We report on the fate of nucleic acids conformation in the gas phase as sampled using native mass spectrometry coupled to ion mobility spectrometry. On the basis of several successful reports for proteins and their complexes, the technique has become popular in structural biology, and the conformation survival becomes more and more taken for granted. Surprisingly, we found that DNA and RNA duplexes, at the electrospray charge states naturally obtained from native solution conditions (≥100 mM aqueous NH4OAc), are significantly more compact in the gas phase compared to the canonical solution structures. The compaction is observed for all duplex sizes (gas-phase structures are more compact than canonical B-helices by ∼20% for 12-bp, and by up to ∼30% for 36-bp duplexes), and for DNA and RNA alike. Molecular modeling (density functional calculations on small helices, semiempirical calculations on up to 12-bp, and molecular dynamics on up to 36-bp duplexes) demonstrates that the compaction is due to phosphate group self-solvation prevailing over Coulomb repulsion. Molecular dynamics simulations starting from solution structures do not reproduce the experimental compaction. To be experimentally relevant, molecular dynamics sampling should reflect the progressive structural rearrangements occurring during desolvation. For nucleic acid duplexes, the compaction observed for low charge states results from novel phosphate–phosphate hydrogen bonds formed across both grooves at the very late stages of electrospray.

QM/MM Studies Reveal How Substrate-Substrate and Enzyme-Substrate Interactions Modulate Retaining Glycosyltransferases Catalysis and Mechanism.

Glycosyltransferases (GTs) catalyze the biosynthesis of glycosidic linkages by transferring a monosaccharide from a nucleotide sugar donor to an acceptor substrate, and they do that with exquisite regio- and stereospecificity. Retaining GTs act with retention of the configuration at the anomeric carbon of the transferred sugar. Their chemical mechanism has been under debate for long as conclusive experimental data to confirm the mechanism have been elusive. In the past years, quantum mechanical/molecular mechanical (QM/MM) calculations have shed light on the mechanistic discussion. Here, we review the work carried out in our group investigating three of these retaining enzymes (LgtC, α3GalT, and GalNAc-T2). Our results support the controversial front-side attack mechanism as the general mechanism for most retaining GTs. The latest structural data are in agreement with these findings. QM/MM calculations have revealed how enzyme-substrate and substrate-substrate interactions modulate the transfer reaction catalyzed by these enzymes. Moreover, they provide an explanation on why in some cases a strong nucleophilic residue is found on the β-face of the sugar, opening the door to a shift toward a double-displacement mechanism.

Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

Significance Nearly 90% of lethal antibiotic-resistant infections in the United States are caused by Gram-positive pathogens, with Staphylococcus aureus accounting for more than one-half of these. Antibiotic resistance is often encoded by plasmids and integrative elements that are exchanged between bacteria through conjugative DNA transfer. During conjugation, a relaxase protein binds, nicks, and covalently attaches to the 5′-end of the DNA, guiding it to the recipient cell, where it restores its circular closed form. We show that relaxase MobM from the promiscuous plasmid pMV158 uses a hitherto unseen mechanism for DNA nicking/closing that is based on the formation of a protein-DNA phosphoramidate adduct. Moreover, our analysis reveals that MobM-like histidine relaxases account for 85% of all relaxases in S. aureus isolates. Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOBV family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOBV relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. We discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOBV histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.

Chapter 7:Molecular Modelling of Nucleic Acids

Nucleic acids (NAs) are biomolecules essential to all known forms of life that exhibit a remarkable structural and functional diversity. NAs are studied through several different techniques, including experimental and theoretical methods. The notorious improvements of the latter, together with the increased computation power, explain their widespread use in improving our understanding of their structure and function. Giving the multiscale nature of NAs, different theoretical disciplines like quantum chemistry, molecular mechanics and mesoscopic biophysical approaches are considered. In that regard, we describe and put into perspective, here, the most recent theoretical methods that have been used to study these biomolecules, from the electronic structure of nucleosides to the structural arrangements of chromosomes.