Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs shows that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. These results are directly applicable to rational enzyme design and engineering.Topics: Enzyme kinetics; Proteins; Quantum mechanics; Quantum mechanics;

Sesquiterpenoids comprise a class of terpenoid natural products with thousands of compounds that are highly diverse in structure, generally containing a polycyclic carbon backbone that is constructed by a sesquiterpene synthase. Decades of experimental and computational studies have demonstrated that these enzymes generate a carbocation in the active site, which undergoes a series of structural rearrangements until a product is formed via deprotonation or nucleophile attack. However, for the vast majority of these enzymes the productive binding orientation of the intermediate carbocations has remained unclear. In this work, a method that combines quantum mechanics and computational docking is used to generate an all-atom model of every putative intermediate formed in the context of the enzyme active site for tobacco epi-aristolochene synthase (TEAS). This method identifies a single pathway that links the first intermediate to the last, enabling us to propose the first high-resolution model for the reaction intermediates in the active site of TEAS, and providing testable predictions.

Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis, an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli. When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project.Keywords: alkane biosynthesis; biofuels; fatty acid biosynthesis; synthetic biology;

The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a kcat/KM of 568 M–1 s–1, representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.

Sesquiterpenoids comprise a class of terpenoid natural products with thousands of compounds that are highly diverse in structure, generally containing a polycyclic carbon backbone that is constructed by a sesquiterpene synthase. Decades of experimental and computational studies have demonstrated that these enzymes generate a carbocation in the active site, which undergoes a series of structural rearrangements until a product is formed via deprotonation or nucleophile attack. However, for the vast majority of these enzymes the productive binding orientation of the intermediate carbocations has remained unclear. In this work, a method that combines quantum mechanics and computational docking is used to generate an all-atom model of every putative intermediate formed in the context of the enzyme active site for tobacco epi-aristolochene synthase (TEAS). This method identifies a single pathway that links the first intermediate to the last, enabling us to propose the first high-resolution model for the reaction intermediates in the active site of TEAS, and providing testable predictions.

Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.