To explore how distal mutations affect binding sites and how binding sites in proteins communicate, an ensemble-based model of the native state was used to define the energetic connectivities between the different structural elements of Escherichia coli dihydrofolate reductase. Analysis of this model protein has allowed us to identify two important aspects of intramolecular communication. First, within a protein, pair-wise couplings exist that define the magnitude and extent to which mutational effects propagate from the point of origin. These pair-wise couplings can be identified from a quantity we define as the residue-specific connectivity. Second, in addition to the pair-wise energetic coupling between residues, there exists functional connectivity, which identifies energetic coupling between entire functional elements (i.e., binding sites) and the rest of the protein. Analysis of the energetic couplings provides access to the thermodynamic domain structure in dihydrofolate reductase as well as the susceptibility of the different regions of the protein to both small-scale (e.g., point mutations) and large-scale perturbations (e.g., binding ligand). The results point toward a view of allosterism and signal transduction wherein perturbations do not necessarily propagate through structure via a series of conformational distortions that extend from one active site to another. Instead, the observed behavior is a manifestation of the distribution of states in the ensemble and how the distribution is affected by the perturbation. M ost cellular processes, which are facilitated by proteins, are modulated by effectors. The basic features of such a mechanism of regulation are the presence of multiple binding sites for various ligands and communication between these binding sites, which often are situated many angstroms apart. An understanding of the ground rules of this regulatory mechanism requires a quantitative definition of the functional linkages between these binding sites. In 1964, Wyman introduced the thermodynamic concept of linked functions to establish a quantitative formulation for describing the mutual influence of binding sites on each other (1). Linkage theory is based on thermodynamic principles, is applicable to all biological systems, and exhibits quantitative predictive power. Although Wyman's theory provides the mathematical relationships, it does not address the mechanism through which different binding sites communicate. Thus, besides functional linkage, there are underlying structural-thermodynamic linkages that define the mechanism of site-site communication.Despite a significant body of literature showing that information is transmitted through biological systems via a series of interand intramolecular communication events (refs. 2-5 and references therein), a quantitative predictive theory of structural linkage analogous to the Wyman Linkage Theory is not available. Recently, however, a theoretical approach was established to treat the native state of a protein as an ensemble of conformationa...
Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the b-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven a-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.
Rose-like odor 2-phenylethanol (2-PE) and its more fruit-like ester 2-phenylethyl acetate (2-PEAc) are two important aromatic compounds and have wide applications. In the past, 2-PE and 2-PEAc were mainly produced from l-phenylalanine. In this study, Escherichia coli was engineered to de novo biosynthesis of 2-PE and 2-PEAc from glucose: first, overexpression of deregulated 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase aroG and chorismate mutase/prephenate dehydratase pheA for increasing phenylpyruvate production in E. coli, subsequently, heterologous expression of decarboxylase kdc and overexpression of reductase yjgB for the conversion of phenylpyruvate to 2-PE, with the engineered strain DG01 producing 578 mg/L 2-PE, and, finally, heterologous expression of an aminotransferase aro8 to redirect the metabolic flux to phenylpyruvate. 2-PE (1016 mg/L) was accumulated in the engineered strain DG02. Alcohol acetyltransferase ATF1 from Saccharomyces cerevisiae can esterify a wide variety of alcohols, including 2-PE. We have further demonstrated the biosynthesis of 2-PEAc from glucose by overexpressing atf1 for the subsequent conversion of 2-PE to 2-PEAc. The engineered strain DG03 produced 687 mg/L 2-PEAc.
Indole-3-acetic acid (IAA) is considered the most common and important naturally occurring auxin in plants and a major regulator of plant growth and development. In this study, an aldehyde dehydrogenase AldH from Escherichia coli was found to convert indole-3-acetylaldehyde into IAA. Then we established an artificial pathway in engineered E. coli for microbial production of IAA from glucose. The overall pathway includes the upstream pathway from glucose to L-tryptophan and the downstream pathway from L-tryptophan to IAA. To our knowledge, this is the first report on the biosynthesis of IAA directly from a renewable carbon source. The study described here shows the way for the development of a beneficial microbe for biosynthesis of auxin and promoting plant growth in the future.
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