Mechanisms For Coenzyme Recognition Of Aldehyde Reductase And Allosteric Regulation Of Aspartokinse

As the environments of organisms are constantly changing, the reactions of metabolism must be finely regulated in order to maintain a constant set of conditions within cells, a condition called homeostasis.Metabolic regulation allows organisms to respond to signals and interact actively with their environments.As essential parts of organisms, proteins participate in virtually every process within cells including metabolic regulation. It is well known that the structure of a protein is the basis for understanding its function. Nevertheless, although static structures are known for many proteins through X-ray crystallography and NMR, the functions of proteins are governed ultimately by their dynamical characters. Therefore, a thorough knowledge of the principles governing protein dynamics is of fundamental importance for functional study and design of new proteins. With the improvement of the theory of quantum mechanics, the update of empirical force field and the increase of computational speed, the theory and methods of molecular simulation have been rapidly developed with applications in physics, chemistry, life science and many other fields. Molecular simulation is able to provide understanding of the basic process of life at the molecular, sub-molecular, atomic, even electronic level.Nowadays, molecular simulation has become an irreplaceable tool in modern bioscience and biotechnology.This dissertation is composed of two main parts.In the first part, by taking the biosynthesis of microbial secondary metabolite of1,3-propanediol as the engineering background, the molecular recognition in regulatory process as the dynamical model and the1,3-propanediol oxireductive reaction as the study object, mechanisms involved in the molecular recognition of1,3-propanediol oxidoreductase and a novel aldehyde reductase for coenzyme I/II were revealed, and the rational design and modification of the coenzyme specificity of1,3-propanediol oxidoreductase was succesfuly accompolished. In the second part, by taking the biosynthesis of microbial primary metabolite L-lysine as the engineering background, the signal transduction in the regulatory process as the dynamical model and the aspartokinase as the study object, a new model which is based on energy dissipation and able to reveal dynamical characters during the process of allesteric regulations was proposed, a novel algorithm for construction of intramolecular signal transduction network was given based on the energy dissipation model, and its application in the analysis of signal transduction process involved in heteromultimeric allosteric proteins was investigated as well. In microbial production of1,3-propanediol, the formation of1,3-propanediol is limited by the amount of NADH supplied by the oxidative pathway of glycerol dismutation. Previous metabolic flux analysis revealed that relaxation of the coenzyme specificity of1,3-propanediol oxidoreductase for both coenzyme I and coenzyme II would increase the production of1,3-propanediol as well as maintaining the NADH-NAD+circle.The work presented in chapter2tries to accomplish such a relaxation by rational protein design. Overall binding free energy indicated that electrostatic energy was the major force discriminating coenzyme Ⅰ from coenzyme Ⅱ.Computational alaninescanning mutagenesis of the active site residues illustrated that Asp41was the key residue responsible for the coenzyme specificity. Compared with Asp41Ala, Asp41Gly could further weaken the repulsion between Asp41and the phosphate group esterified to the2'-hydroxyl group of the ribose at the adenine end of coenzyme Ⅱ. Site-directed mutagenesis was then conducted and the relaxation was successfully realized.During the fermentative production of1,3-propanediol under high substrate concentrations, accumulation of intracellular3-hydroxypropionaldehyde will cause premature cessation of cell growth and glycerol consumption. Discovery of oxidoreductases that can convert3-hydroxypropionaldehyde to1,3-propanediol using coenzyme Ⅱ as cofactor could serve as a solution to this problem.In chapter3, a novel aldehyde reductase from Klebsiella pneumoniae DSM2026was cloned and characterized.The aldehyde reductase showed broad substrate specificity in the physiological direction, whereas no catalytic activity was detected in the oxidation direction. Furthermore, both coenzyme Ⅰ and coenzyme Ⅱ could be utilized as cofactor. The cofactor binding mechanism was then investigated employing homology modeling and molecular dynamics simulations.Hydrogen-bond analysis showed that the hydrogen-bond interactions between the aldehyde reductase and coenzyme Ⅱ were much stronger than that for coenzyme Ⅰ.Free energy decomposition dedicated that residues of Gly37to Val41contributed most to the cofactor preference through polar interactions.This work provides a novel aldehyde reductase that has potential applications in the development of novel genetically engineered strains in1,3-propanediol industry as well as giving a better understanding of the mechanisms involved in cofactor binding.Protein dynamics is essential for its function, especially for intramolecular signal transduction. In chapter4we proposed a new concept based on energy dissipation to systematically reveal protein dynamics upon effector binding and energy perturbation. The concept was applied to better understand the intramolecular signal transduction during allostery of enzymes.The E. coli allosteric enzyme, aspartokinase Ⅲ, was used as a model system and special molecular dynamics simulations were designed and carried out. Computational results indicated that the number of residues affected by external energy perturbation (i.e.caused by a ligand binding) during the energy dissipation process showed a sigmoid pattern. Using the two-state Boltzmann equation, we defined two parameters, the half response time and the dissipation rate constant, which could be used to well characterize the energy dissipation process.For the allostery of aspartokinase III, the residue response time indicated that besides the ACT2signal transduction pathway, there was another pathway between the regulatory site and the catalytic site, which was suggested to be the (315-aK loop of ACT1.We further introduced the term'protein dynamical modules'based on the residue response time.Different from the protein structural modules which merely provide information about the structural stability of proteins, protein dynamical modules could reveal protein characteristics from the perspective of dynamics.Finally, the energy dissipation model was applied to investigate E. coli aspartokinase III mutations to better understand the desensitization of product feedback inhibition via allostery.A novel approach to reveal intramolecular signal transduction network was proposed in chapter5.To this end, a new algorithm of network construction was developed, which was based on the new protein dynamics model of energy dissipation proposed in chapter4.A key feature of this approach was that direction information was specified after inferring protein residue-residue interaction network involved in the process of signal transduction. This enables fundamental analysis of the regulation hierarchy and identification of regulation hubs of the signaling network.The well-studied allosteric enzyme, E. coli aspartokinase III, was used as a model system to demonstrate the new method. Comparison with experimental results showed that the new approach was able to predict all the mutation sites that have been experimentally proved to desensitize allosteric regulation of the enzyme.In addition, the signal transduction network showed a clear preference for specific structural regions, secondary structural types and residue conservation. Occurrence of super-hubs in the network indicated that allosteric regulation tends to gather residues with high connection ability to collectively facilitate the signaling process.Furthermore,a new parameter of propagation coefficient was defined to determine the propagation capability of residues within a signal transduction network.Dynamical intersubunit interactions are key elements in the regulation of many biological systems.Better understanding of how subunits interact with each other and how their interactions are related to dynamic protein structure is a fundamental task in biology. In the previous chapter, a new algorithm of network construction was developed, which was based on the new protein dynamics model of energy dissipation. In chapter6, a heteromultimeric allosteric protein, Corynebacterium glutamicum aspartokinse, was used as a model system to explore signal transduction involved in intersubunit interactions and allosteric communication with an emphasis on the intersubunit signaling process.For this purpose, energy dissipation simulation and network construction were conducted for each subunit and the whole protein. Comparison with experimental results showed that the new approach was able to predict all the mutation sites that have been experimentally proved to desensitize allosteric regulation of the enzyme.Additionally, the analysis revealed that the function of the effector threonine was to facilitate the binding of the two subunits without contributing to the allosteric communication. During the allosteric regulation upon the binding of the effector lysine, signals could be transferred from the β-subunit to the catalytic site of the a-subunit through both a direct way of intersubunit signal transduction, and an indirect way:first to the regulatory region of the a-subunit by intersubunit signal transduction and then to the catalytic region by intramolecular signal transduction. The new approach is not only able to illustrate the diversity of the underlying mechanisms when the strength of feedback inhibition by the product(s) is reduced but also able to provide useful information that has potential applications in engineering heteromultimeric allosteric regulation.In summary, by taking molecular recognition and signal transduction involved in metabolic regulation as examples, this dissertation revealed the mechanisms underlying protein dynamical behaviors during the process of metabolic regulation, providing both theoretical basis and methodologies for the application of molecular engineering of proteins in the field of metabolic regulation.