Structure-function studies of serine hydrolases: synthesis of a gene for ∝-lytic protease and the purification and characterization of a mutant β-lactamase

The author has constructed a synthetic gene for ∝-lytic protease. Since the DNA sequence of the protein is not known, the gene was designed by using the reverse translation of ∝-lytic protease's amino acid sequence. Unique restriction sites are carefully sought in the degenerate DNA sequence to aid in future mutagenesis studies. The unique restriction sites are designed approximately 50 base pairs apart and their appropriate codons used in the DNA sequence. The codons used to construct the DNA sequence of ∝-lytic protease are preferred codons in E-coli or used in the production of β-lactamase. Codon usage is also distributed evenly to ensure that one particular codon is not heavily used. The gene is essentially constructed from the outside in. The gene is built in a stepwise fashion using plasmids as the vehicles for the ∝-lytic oligomers. The use of plasmids allows the replication and isolation of large quantities of the intermediates during gene synthesis. The ∝-lytic DNA is a double-stranded oligomer that has sufficient overhang and sticky ends to anneal correctly in the vector. After six steps of incorporating ∝-lytic DNA, the gene is completed and sequenced to ensure that the correct DNA sequence is present and that no mutations occurred in the structural gene.

β-lactamase is the other serine hydrolase studied in this thesis. The author used the class A RTEM-1 β- lactamase encoded on the plasmid pBR322 to investigate the roll of the conserved threonine residue at position 71. Cassette mutagenesis was previously used to generate all possible amino acid substitutions at position 71. The work presented here describes the purification and kinetic characterization of a T71H mutant previously constructed by S. Schultz. The mutated gene was transferred into plasmid pJN for expression and induced with IPTG. The enzyme is purified by column chromatography and FPLC to homogeneity. Kinetic studies reveal that the mutant has lower k_(cat) values on benzylpenicillin, cephalothin and 6-aminopenicillanic acid but no changes in k_m except for cephalothin which is approximately 4 times higher. The mutant did not change siginificantly in its pH profile compared to the wild-type enzyme. Also, the mutant is more sensitive to thermal denaturation as compared to the wild-type enzyme. However, experimental evidence indicates that the probable generation of a positive charge at position 71 thermally stabilized the mutant.

Olefin cyclopropanation and carbon-hydrogen amination via carbene and nitrene transfers catalyzed by engineered cytochrome P450 enzymes

Synthetic biology promises to transform organic synthesis by enabling artificial catalysis in living cells. I start by reviewing the state of the art in this young field and recognizing that new approaches are required for designing enzymes that catalyze nonnatural reactions, in order to expand the scope of biocatalytic transformations. Carbene and nitrene transfers to C=C and C-H bonds are reactions of tremendous synthetic utility that lack biological counterparts. I show that various heme proteins, including cytochrome P450BM3, will catalyze promiscuous levels of olefin cyclopropanation when provided with the appropriate synthetic reagents (e.g., diazoesters and styrene). Only a few amino acid substitutions are required to install synthetically useful levels of stereoselective cyclopropanation activity in P450BM3. Understanding that the ferrous-heme is the active species for catalysis and that the artificial reagents are unable to induce a spin-shift-dependent increase in the redox potential of the ferric P450, I design a high-potential serine-heme ligated P450 (P411) that can efficiently catalyze cyclopropanation using NAD(P)H. Intact E. coli whole-cells expressing P411 are highly efficient asymmetric catalysts for olefin cyclopropanation. I also show that engineered P450s can catalyze intramolecular amination of benzylic C-H bonds from arylsulfonyl azides. Finally, I review other examples of where synthetic reagents have been used to drive the evolution of novel enzymatic activity in the environment and in the laboratory. I invoke preadaptation to explain these observations and propose that other man-invented reactions may also be transferrable to natural enzymes by using a mechanism-based approach for choosing the enzymes and the reagents. Overall, this work shows that existing enzymes can be readily adapted for catalysis of synthetically important reactions not previously observed in nature.

Engineering thermostable fungal cellobiohydrolases

Meeting the world's growing energy demands while protecting our fragile environment is a challenging issue. Second generation biofuels are liquid fuels like long-chain alcohols produced from lignocellulosic biomass. To reduce the cost of biofuel production, we engineered fungal family 6 cellobiohydrolases (Cel6A) for enhanced thermostability using random mutagenesis and recombination of beneficial mutations. During long-time hydrolysis, engineered thermostable cellulases hydrolyze more sugars than wild-type Cel6A as single enzymes and binary mixtures at their respective optimum temperatures. Engineered thermostable cellulases exhibit synergy in binary mixtures similar to wild-type cellulases, demonstrating the utility of engineering individual cellulases to produce novel thermostable mixtures. Crystal structures of the engineered thermostable cellulases indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by proline substitutions. At high temperature, free cysteines contribute to irreversible thermal inactivation in engineered thermostable Cel6A and wild-type Cel6A. The mechanism of thermal inactivation in this cellulase family is consistent with disulfide bond degradation and thiol-disulfide exchange. Enhancing the thermostability of Cel6A also increases tolerance to pretreatment chemicals, demonstrated by the strong correlation between thermostability and tolerance to 1-ethyl-3-methylimidazolium acetate. Several semi-rational protein engineering approaches – on the basis of consensus sequence analysis, proline stabilization, FoldX energy calculation, and high B-factors – were evaluated to further enhance the thermostability of Cel6A.

Electrochemical and electron transfer investigations of copper proteins

A study of the pH and temperature dependence of the redox potentials of azurins from five species of bacteria has been performed. The variations in the potentials with pH have been interpreted in terms of electrostatic interactions between the copper site and titrating histidine residues, including the effects of substitutions in the amino acid sequences of the proteins on the electrostatic interactions. A comparison of the observed pH dependences with predictions based on histidine pK_a values known for Pseudomonas aeruginosa (Pae), Alcaligenes denitrificans (Ade), and Alcaligenes faecalis (Afa) azurins indicates that the Pae and Ade redox potentials exhibit pH dependences in line with electrostatic arguments, while Afa azurin exhibits more complex behavior. Redox enthalpies and entropies for four of the azurins at low and high pH values have also been obtained. Based on these results in conjuction with the variable pH experiments, it appears that Bordetella bronchiseptica azurin may undergo a more substantial conformational change with pH than has been observed for other species of azurin.

The temperature dependence of the redox potential of bovine erythrocyte superoxide dismutase (SOD) has been determined at pH 7.0, with potassium ferricyanide as the mediator. The following thermodynamic parameters have been obtained (T = 25°C): E°' = 403±5 mV vs. NHE, ΔG°' = -9.31 kcal/mol, ΔH°' = -21.4 kcal/mol, ΔS°' = -40.7 eu, ΔS°'_(rc) = -25.1 eu. It is apparent from these results that ΔH°', rather than ΔS°', is the dominant factor in establishing the high redox potential of SOD. The large negative enthalpy of reduction may also reflect the factors which give SOD its high specificity toward reduction and oxidation by superoxide.

Structural Characterization of Pro-inflammatory and Anti-inflammatory Immunoglobulin G Fc Proteins

Immunoglobulin G (IgG) is central in mediating host defense due to its ability to target and eliminate invading pathogens. The fragment antigen binding (Fab) regions are responsible for antigen recognition; however the effector responses are encoded on the Fc region of IgG. IgG Fc displays considerable glycan heterogeneity, accounting for its complex effector functions of inflammation, modulation and immune suppression. Intravenous immunoglobulin G (IVIG) is pooled serum IgG from multiple donors and is used to treat individuals with autoimmune and inflammatory disorders such as rheumatoid arthritis and Kawasaki’s disease, respectively. It contains all the subtypes of IgG (IgG1-4) and over 120 glycovariants due to variation of an Asparagine 297-linked glycan on the Fc. The species identified as the activating component of IVIG is sialylated IgG Fc. Comparisons of wild type Fc and sialylated Fc X-ray crystal structures suggests that sialylation causes an increase in conformational flexibility, which may be important for its anti-inflammatory properties.

Although glycan modifications can promote the anti-inflammatory properties of the Fc, there are amino acid substitutions that cause Fcs to initiate an enhanced immune response. Mutations in the Fc can cause up to a 100-fold increase in binding affinity to activating Fc gamma receptors located on immune cells, and have been shown to enhance antibody dependent cell-mediated cytotoxicity. This is important in developing therapeutic antibodies against cancer and infectious diseases. Structural studies of mutant Fcs in complex with activating receptors gave insight into new protein-protein interactions that lead to an enhanced binding affinity.

Together these studies show how dynamic and diverse the Fc region is and how both protein and carbohydrate modifications can alter structure, leading to IgG Fc’s switch from a pro-inflammatory to an anti-inflammatory protein.

I. Efforts toward the synthesis of aliphatic iodonium salts. II. Flourine-19 nuclear magnetic resonance spectroscopy of cyclic and bicyclic systems

The synthesis of iodonium salts of the general formula [C₆H₅IR]⁺X^-, where R is an alkyl group and x- is a stabilizing anion, was attempted. For the choice of R three groups were selected, whose derivatives are known to be sluggish in S_N1 and S_N2 substitutions: cyclopropyl, 7, 7 -dimethyl-1-norbornyl, and 9 -triptycyl. The synthetic routes followed along classical lines which have been exploited in recent years by Beringer and students. Ultimately, the object of the present study was to study the reactions of the above salts with nucleophiles. In none of the three cases, however, was it possible to isolate a stable salt. A thermodynamic argument suggests that this must be due to kinetic instability rather than thermodynamic instability. Only iodocyclopropane and 1-iodoapocamphane formed isolable iododichlorides.

Several methylated 2, 2-difluoronorbornanes were prepared with the intent of correlating fluorine -19 chemical shifts with geometric features in a rigid system. The effect of a methyl group on the shielding of a β -fluorine is dependent upon the dihedral angle; the maximum effect (an upfield shift of the resonance) occurs at 0° and 180°, whereas almost no effect is felt at a dihedral angle of 120°. The effect of a methyl group on a γ -fluorine is to strongly shift the resonance downfield when fluorine and methyl group are in a 1, 3 - diaxial-like relationship. Molecular orbital calculations of fluorine shielding in a variety of molecules were carried out using the formalism developed by Pople; the results are, at best, in modest agreement with experiment.

Large plane deformations of thin elastic sheets of neo-hookean material

Large plane deformations of thin elastic sheets of neo-Hookean material are considered and a method of successive substitutions is developed to solve problems within the two-dimensional theory of finite plane stress. The first approximation is determined by linear boundary value problems on two harmonic functions, and it is approached asymptotically at very large extensions in the plane of the sheet. The second and higher approximations are obtained by solving Poisson equations. The method requires modification when the membrane has a traction-free edge.

Several problems are treated involving infinite sheets under uniform biaxial stretching at infinity. First approximations are obtained when a circular or elliptic inclusion is present and when the sheet has a circular or elliptic hole, including the limiting cases of a line inclusion and a straight crack or slit. Good agreement with exact solutions is found for circularly symmetric deformations. Other examples discuss the stretching of a short wide strip, the deformation near a boundary corner which is traction-free, and the application of a concentrated load to a boundary point.