Browsing by Subject "Enzyme"

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Application of enzymatic catalysis and galvanic processes for biosensor development
(2011-08) Zaccheo, Brian Andrew; Crooks, Richard M. (Richard McConnell); Browning, Karen; Hoffman, David; Johnston, Keith P.; Stevenson, Keith
Methods for integrating enzyme systems with electrochemical reactions having applications to diagnostic sensing are described. Diagnostic tests that include biological molecules can be classified as biosensors. Existing testing methods often require trained technicians to perform, and laboratory settings with complex infrastructure. The theme of this dissertation is the development of methods that are faster, easier to use, and more applicable for non-laboratory environments. These goals are accomplished in systems using enzymatic catalysis and galvanic processes. Two biosensors with specific model pathologies have been designed and demonstrated in this study. The first assay senses a DNA fragment representing the Epstein Barr virus and uses enzyme-mediated Ag deposition over a v microfabricated chip. The chip contains a specially designed pair of electrodes in an interdigitated array (IDA). Detection is signaled by a change in the resistance between the two electrodes. The second biosensor discussed in this study is targeted towards the digestive enzyme trypsin. It is selfpowered due to its construction within an open-circuit galvanic cell. In this system, a small volume of blood serum is introduced onto the device over barriers made of protein and Al that block the anode from solution. In the presence of trypsin, the protein gel is rendered more permeable to sodium hydroxide. Adding hydroxide initiates the dissolution of the Al layer, closing the cell circuit and illuminating a light-emitting diode (LED). A relationship was observed between LED illumination time and trypsin concentration. Biosensors that utilize enzymes to generate or amplify a detectable signal are widely used, and the final project of this study uses a nanoparticle based approach to protect the catalytic activity of alkaline phosphatase (AlkP) from hostile chemicals. By incubating Au colloid with AlkP overnight and adding Ag+, core@shell nanoparticles of Au@Ag2O can be isolated that show AlkP activity. The resulting enzyme-metal composite material was analytically characterized and demonstrated greater activity in the presence of organic inhibitors relative to either wild type vi or Au colloid-associated AlkP without the Ag2O shell. The stabilization procedure is complete in one day using a onepot synthesis. This method may provide opportunities to carry out biosensing chemistry in previously incompatible chemical environments.
Biosynthetic studies of thiosugar natural products ; Mechanistic studies of hyoscyamine 6 [beta]-hydroxylase
(2023-01-02) Ushimaru, Richiro; Liu, Hung-wen, 1952-; Krische, Michael J; Martin, Stephen F; Whitman, Christian P; Keatinge-Clay, Adrian
Natural products produced by microorganisms and plants provide a rich source of novel and diverse chemical entities. Understanding the biosynthetic pathways of these natural products and the chemical mechanisms of the enzymes involved has been essential for modern drug discovery and development. This dissertation describes studies on the biosynthetic pathways of natural products with atypical chemical motifs. Part I describes biosynthetic studies of the lincosamides and albomycins, which are two groups of thiosugar-containing natural products. Lincosamides such as lincomycin A and celesticetin share a thioglycoside core linked to a proline residue but contain different substituents on the sulfur center at C1. It will be demonstrated that three stand-alone nonribosomal peptide synthetase domains are responsible for conjugation between the proline residue and the thiosugar core. In addition, the pyridoxal 5′- phosphate-dependent enzymes LmbF and CcbF are shown to be key enzymes that lead to bifurcation of the lincosamide biosynthetic pathway and differential modification of lincomycin A versus celesticetin. Albomycins are 4′-thiofuranose-containing nucleosides that are conjugated with a ferrichrome-type siderophore through a serine linker residue. It will be shown that AbmM catalyzes formation of the sulfur-bridged furanose core via a radical-mediated mechanism. In addition, it will also be demonstrated that the resulting 4′-thiofuranosyl nucleotide is matured into SB-217452, which is the active antibiotic component of albomycins, via reactions catalyzed by AbmG, AbmH, AbmD, AbmK, AbmF, and AbmJ. Part II describes mechanistic studies of hyoscyamine 6β-hydroxylase (H6H). H6H catalyzes hydroxylation and subsequent oxidative cyclization to form an epoxide ring from an ethylene group during scopolamine alkaloid biosynthesis. Studies using substrate analogues indicate that substrate conformation correlates with selectivity between the dihydroxylation and epoxidation activities of H6H. Based on kinetic analysis, it will also be shown that the presence of a hydroxyl group in the substrate significantly affects the regioselectivity of H atom abstraction during H6H-catalyzed oxidation reactions.
Characterization of two radical S-adenosyl-L-methionine enzymes in the biosynthesis of aminoglycosides
(2016-05-10) Kim, Hak Joong, active 21st century,; Liu, Hung-wen, 1952-; Anslyn, Eric V; Dong, Guangbin; Keatinge-Clay, Adrian T; Whitman, Christian P
Biosynthetic studies of natural products are essential to the discovery and development of new drugs, because by understanding biosynthetic pathways and the enzymes that characterize them, new pathways can be engineered for the production of new compounds with improved clinical properties. Aminoglycosides have traditionally been used as important antibiotics, but resistance against aminoglycosides is well known. This has resulted in a need to better understand the biosynthesis of aminoglycosides. Besides the practical value, the discovery of new enzymes with unprecedented functions in the biosynthesis of aminoglycosides can expand our knowledge and advance our understanding of enzyme catalysis. The work described in this dissertation focuses on the in vitro characterization of two radical S-adenosyl-L-methionine (SAM) enzymes in the biosynthesis of aminoglycosides. First, GenK is a cobalamin (Cbl)-dependent radical SAM enzyme that is responsible for catalyzing the methylation of gentamicin X₂ to produce G418. In vitro assays of purified and reconstituted GenK from Micromonospora echinospora showed that this enzyme is a radical SAM enzyme with one [4Fe-4S] cluster. Assays of GenK with SAM, gentamicin X₂ and Cbl confirmed that the methylation reaction occurs at an unactivated carbon during gentamicin biosynthesis. Isotope labeling experiments strongly suggested that SAM is the preliminary methyl donor to cobalamin, followed by secondary transfer from Me-Cbl to gentamicin X₂. It was demonstrated that GenK also accepts alternative aminoglycoside substrates besides gentamicin X₂. Second, three possible mechanisms for the GenK reaction were suggested and tested. Experiments investigating the stoichiometry of the GenK reaction revealed 5'-deoxyadenosine (5'-dAdo), S-adenoxylhomocysteine (SAH), and G418 were produced in equal proportion and one-to-one with each methylation reaction catalyzed by GenK. The experiment with labeled substrates indicated that the 6'-pro-R-hydrogen atom of gentamicin X₂ is abstracted by 5'-dAdo• and that methylation occurs with retention of configuration at C6'. Several substrate derivatives were synthesized to investigate the manner of methyl transfer from cobalamin to the substrate. Finally, the in vitro characterization of AprD4 and AprD3 in the biosynthesis of C3'-deoxyaminoglycoside was also conducted. Purified and reconstituted AprD4 from Streptomyces tenebrarius is a radical SAM enzyme, catalyzing homolysis of SAM to 5'-dAdo in the presence of paromamine to produce 4-oxolividamine. AprD3 from the same strain is shown to be a dehydrogenase acting as the reductase counterpart to AprD4 to catalyze the reduction of 4-oxolividamine to generate lividamine. The experiments with labeled compounds confirmed the regiochemistry of hydrogen atom abstraction by 5'-dAdo• and the stereochemical course of C3'-deoxygenation of paromamine.
Enzymatic treatment of pharmaceuticals and personal care products (PPCPs) in municipal wastewater
(2013-05) Sharkey, Margaret E; Lawler, Desmond F.; Kinney, Kerry A.
Conventional wastewater treatment plants do not effectively remove pharmaceuticals and personal care products (PPCPs). As a result, PPCPs enter the environment via treated wastewater discharge. Enzymatic treatment, using the laccasemediator system, is a novel biochemical process that has been shown to effectively treat some PPCPs. This study investigates the efficacy of the laccase-mediator system to treat PPCPs using a process that can be easily implemented at an existing wastewater treatment plant. Enzymatic treatment will be most beneficial after primary sedimentation and before conventional biological treatment, where unoxidized PPCPs and byproducts could have the opportunity for further degradation in biological treatment. In this work, two enzymatic treatment configurations were studied. A step-wise optimization process was used that alternately varied treatment conditions: pH, enzyme activity, mediator concentration, and reactor detention time. In the optimization process of each configuration, successful oxybenzone removal (~90%) was achieved in municipal primary effluent. In a direct comparison of treatment configurations, both resulted in vi similar percent removals of oxybenzone. Therefore, the configuration with the simpler operation and reactor design was chosen for further study. During the optimization process, several noteworthy conclusions were made that might have full-scale enzymatic treatment implications. Specifically, successful oxybenzone removal occurred at unadjusted pH and without aeration, but increased biological oxygen demand of the wastewater increased the required mediator concentration. While the first finding would decrease enzymatic treatment costs, the latter would increase the costs associated with the mediator. Thus, an alternative mediator source, specifically one high in phenolic compounds, is desired. The use of wine, as a surrogate of winery wastewater, was in investigated and proved ineffective. Further investigation of alternative mediator sources is required. Treatment of another PPCP, sulfamethoxazole, was less efficient (65% removal) than that of oxybenzone, but nevertheless, the substantial removal might indicate that other PPCPs can be treated with the laccase-mediator system. The most promising result of this work was the simultaneous treatment of multiple PPCPs, oxybenzone and sulfamethoxazole. Simultaneous treatment proved to be as effective as when each PPCP was treated individually.
Enzyme immobilization on gold surfaces : effects of surface chemistry and attachment strategies on binding and activity
(2023-04-20) Correira, Joshua Manuel; Webb, Lauren J.; Samanta, Devleena; Baiz, Carlos R; Gordon, Vernita; Shear, Jason B
Functional enzymes are the basis for many biotechnological systems, including biosensors, bio-fuel cells, and heterogeneous biocatalysts. In these systems, enzymes are often immobilized on a solid support or surface to capture their catalytic activity. Immobilization has the advantage of improving enzyme stability and reusability but often results in a significant loss of catalytic activity (1-2 orders of magnitude) when compared to the native enzyme. This inactivation is due to direct interactions between the enzyme and the solid support. Here, we developed methods to study enzyme immobilization and resulting inactivation. The aim of this work was to identify optimal surface chemistries and attachment strategies that promote high binding efficiencies while minimizing activity losses. Subsequently, we studied this using three enzymes (acetylcholinesterase (AChE), β-galactosidase (β-gal), horseradish peroxidase (HRP)) immobilized on gold surfaces by direct adsorption, covalent attachment, and DNA-directed attachment. First, AChE was directly adsorbed onto a variety of gold surfaces modified with self-assembled monolayers (SAMs) terminated with -COO⁻, -NH₃⁺, -OH, and -CH₃ functional groups at varying mole % to study the effect of surface hydrophobicity and charge on binding and activity. We found that binding was directly proportional to surface hydrophobicity (r = 0.75) and activity was inversely proportional to surface hydrophobicity (r = -0.62). The highest binding observed was ~40% of a monolayer on the most hydrophobic surfaces and the lowest binding observed was ~10% of a monolayer on the most hydrophilic surfaces. Conversely, on the most hydrophobic surfaces AChE retained <10% of its native activity, and on the most hydrophilic surfaces AChE retained ~40% of its native activity. This illuminated an inherent problem with direct adsorption: high binding and high activity are mutually exclusive. Due to these findings, we next immobilized β-gal and HRP on DNA-functionalized gold surfaces using DNA-DNA interactions, to avoid direct interactions between the enzyme and surface. We found that β-gal retained 62% of its native activity following immobilization, a significant improvement over previous direct adsorption strategies.
Evolved enzymes for cancer therapeutics and orthogonal systems
(2013-08) Lu, Wei-Cheng; Ellington, Andrew D.
Directed evolution has been explored for a long time. Various ideas, methods, have been shown to be feasible and successful in the enzyme field. We were interested in evolving enzymes for applications. Therefore, we evolved human cystathionine gamma-lyase (hCGL) and E. coli biotin ligase for therapeutic and biotechnology applications. Wild-type human cystathionine gamma-lyase does not have any methionine-degrading activity, unlike the high methionine-degrading abilities of bacterial methionine gamma-lyase (MGL) found in Pseudomonas putida. The ability to engineer hCGL to breakdown methionine can be a potential cancer treatment by targeting the methionine-dependent cancer cells. However, the methionine-degrading activity of previously engineered hCGL has only shown 1% activity compared to MGL, too low to be useful in practical cancer therapeutics. By using a combination of protein design and phylogenetic analysis, we further evolved hCGL to achieve a higher methionine-degrading activity, with one variant displaying as much as 7% activity compared to bacterial MGL, making it a more likely candidate in cancer treatment.In addition, it has been shown that new orthogonal pairs of biotin protein ligase and biotin have many biotechnology applications. Therefore, we have developed selection scheme for directing the evolution of E. coli biotin protein ligase (BPL, gene: BirA) via in vitro compartmentalization, and have altered the substrate specificity of BPL towards the utilization of the biotin analogue desthiobiotin. Following just 6 rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA₆₋₄₀ and BirA₆₋₄₇, showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. Further characterization of BirA₆₋₄₀ and the single substitution variant BirA[subscript M157T] revealed that they had 2- to 3-fold higher kcat values for desthiobiotin, and 3- to 4-fold higher K[subscript M] values. The k[subscript cat]/K[subscript M] values for these enzymes were around 0.7-fold that of BirA[subscript wt-]. It is interesting the selections did not lower the K[subscript M] for desthiobiotin and actually led to a less efficient enzyme. This is an example of how "you get what you select for". Because peptide:DNA conjugates were distributed such that there was on average one template or less per emulsion compartment there was selection only for the catalytic rate (k[subscript cat]) of desthiobiotinylation and not for turnover. Given these conditions, it might be anticipated that the peptide substrate, rather than desthiobiotin, should be bound better by the winning variants, and in fact BirA₆₋₄₀ showed a reduced K[subscript M] value for BAP.
Novel strategies towards engineering therapeutic enzymes with reduced immunogenicity for cancer therapy
(2010-12) Cantor, Jason Robert; Georgiou, George; Iverson, Brent L.; Maynard, Jennifer A.; Mullins, Charles B.; Whitman, Christian P.
Heterologous enzymes have been investigated for a variety of therapeutic applications, including the treatment of a number of cancers that are sensitive to the systemic depletion of specific amino acids. One such example is acute lymphoblastic leukemia (ALL) for which enzyme-mediated L-Asparagine (L-Asn) depletion by the Escherichia coli L-Asparaginase II (EcAII) has been proven critical for treatment. However, the repeated or prolonged therapeutic administration of such enzymes is restricted by their immunogenicity, which frequently results in the generation of anti-enzyme antibodies that may in turn mediate a variety of adverse hypersensitivity reactions and neutralization of the enzymes themselves. Thus, while the therapeutic efficacy of asparaginase is well established, a significant number of patients still develop adverse immune responses to the enzyme. Here, we have developed and explored novel strategies towards engineering an asparaginase with reduced immunogenicity for ALL therapy. First, we identified and investigated human enzymes that putatively shared functional similarity to asparaginase with the long-term aim of engineering such enzymes to acquire biochemical and pharmacological properties requisite for eventual therapeutic application. In one study, we described the bacterial expression and characterization of the human asparaginase-like protein 1 (hASRGL1). We presented evidence that hASRGL1 exhibited an activity profile consistent with enzymes previously designated as [Beta]-aspartyl peptidases, which had only been previously identified in plants and bacteria. Similar to non-mammalian [Beta]-aspartyl peptidases, hASRGL1 was revealed to be an N-terminal nucleophile (Ntn) hydrolase whereby Thr168 serves as the essential Ntn for both intramolecular processing and catalysis. In a second study, we described the optimized bacterial expression and biochemical characterization of the human N-terminal asparagine amidohydrolase 1 (hNTAN1). We demonstrated that hNTAN1 catalysis is dependent upon direct involvement of a thiol group, and subsequently identified Cys75 as an essential residue that may act as the catalytic nucleophile. Further, we presented the first description of hNTAN1 kinetics, secondary structure composition, and thermal stability. Second, we devised and validated a novel therapeutic deimmunization approach by combinatorial T-cell epitope removal using neutral drift. We showed that combinatorial saturation mutagenesis coupled with a robust neutral drift screen enabled the isolation of engineered EcAII variants that contained multiple amino acid substitutions yet exhibited catalytic efficiencies nearly indistinguishable to that of the parent enzyme. Three regions of EcAII were computationally identified as putative T-cell epitopes and then subjected to saturation mutagenesis at 4 positions (per region) believed to be critical for MHC-II binding. The resulting libraries were then sequentially subjected to a neutral drift FACS screen in order to isolate EcAII mutants that retained wild-type function. Pools of neutral drift variants were then computationally evaluated for MHC-II binding and those that displayed scores indicative of compromised binding were purified and biochemically characterized. Finally, T-cell activation assays and antibody titers in HLA-transgenic mice were used to evaluate T-cell epitope removal and immunogenicity, respectively. Ultimately, we revealed that mice immunized with an EcAII neutral variant containing 8 amino acid substitutions -- 3 of which were non-phylogenetically conserved -- within computationally predicted T-cell epitopes, displayed a significant 10-fold reduction in serum anti-EcAII IgG titer relative to mice similarly immunized with the parent enzyme.
Old dog, new tricks : repurposing iron-carbide-carbonyl clusters as precursors for structural modeling of the nitrogenase cofactor
(2020-05-08) Joseph, Christopher, Ph. D.; Rose, Michael J., Ph. D.; Humphrey, Simon M; Jones, Richard A; Anslyn, Eric V; Milliron, Delia J
Nitrogenases are the only known biological enzyme capable of catalyzing the transformation of dinitrogen (N₂) into ammonia (NH₃). The active site of nitrogenase is comprised of a double-cuboidal iron-sulfur cluster featuring an interstitial carbide as the shared vertex, three ‘belt’ sulfides bridging the cuboidal components, and either a homocitrate-bearing heterometal (Mo, V) or an Fe at one of the distal capping metal sites. Out of the three nitrogenases, the Mo-dependent variant demonstrates the highest activity for N₂ conversion. The active-site cofactor of Mo-dependent nitrogenase (FeMoco) was first isolated in 1977; however, after decades of kinetic, structural, and spectroscopic research, many questions surrounding the mechanism of substrate reduction and the electronic structure of reaction intermediates remain unanswered. In this regard, the synthetic modelling community has contributed significantly towards directing mechanistic discussions with N₂-reducing functional model compounds. Furthermore, structural model compounds have played a pivotal role in deciphering the structural and electronic properties of FeMoco, including the identification of the central carbide and assignment of metal-site valence and spin states. Despite this remarkable progress, a synthetic model featuring a paramagnetic iron cluster with sulfides, interstitial carbide, and heterometal Mo has yet to be reported. The work relayed in this dissertation outlines our efforts towards pursuing this synthetic goal. As such, we utilize a family of carbonyl-supported iron clusters — first reported in the 1960s — featuring iron-coordinated inorganic carbide. However, the highly symmetric packing structures have made heterometal-containing carbidocarbonyl iron clusters difficult to unambiguously characterize by X-ray crystallography. Moreover, the strongly π-acidic ligation sphere enforces low metal-valance states and overall diamagnetism, and ligand substitution of COs is difficult to control. Here, we demonstrate a strategy to disrupt the symmetry in molybdenum-containing heteroclusters to unambiguously characterize the Mo site in XRD. Additionally, CO→S ligand substitution is achieved with the utilization of electrophilic sulfur sources, leading to progressively higher oxidation state Fe sites. These synthetic approaches for heterometal incorporation and oxidative sulfur insertion will serve as fundamental stepping-stones towards future endeavors in utilizing and functionalizing carbidocarbonyl iron clusters as synthetic precursors and ultimately, in biomimetically modeling the nitrogenase active site cluster.
Structure-function analysis of a Group II Intron reverse transcriptase
(2022-05-09) Alvarado Torres, Jose Mario; Lambowitz, Alan; Russell, Rick
Mobile group II introns are ribozymes and mobile genetic elements found in all branches of life. They consist of a highly structured intron RNA that encodes a reverse transcriptase (RT). This RT is utilized during retrohoming to synthesize a full-length DNA copy (cDNA) of the intron RNA which is then integrated into new genome locations. One theory suggests that RTs evolved from an RNA Dependent RNA Polymerase (RDRP) that originated in the RNA World. These ancestral RDRPs served to propagate genetic material, and during the transition to DNA-based life, RDRPs evolved into RTs. Recent structural data for Geobacillus stearothermophilus Group II Intron RT (GsI-IIC RT) suggests a close evolutionary relationship between RDRPs and group II intron RTs, including a strikingly similar RT active site and regions that function in binding the template and primer. To further probe this evolutionary relationship rational mutations were introduced into GsI-IIC RT that affected rNTP incorporation ability. Group II introns have also potentially evolved characteristics like higher fidelity and processivity compared to other RTs due to stringent accuracy requirements when reverse transcribing the catalytically active and highly structured intron RNA. Host-encoded group II intron-like RTs that have acquired other functions often have substitutions for the conserved A residue in the YADD motif at the RT active site. This suggests that the active site can be modified by evolutionary pressures to suit diverse activities. This tolerance was exploited to mutate the highly conserved YADD motif to study structure-function relationships between conserved features of previously uncharacterized classes of RTs. Current laboratory data suggests that the YXDD motif can be modified to modulate different activities, although other crucial complementary or compensatory mutations are likely necessary. Through this work I obtained data that suggests group II intron RTs evolved from ancestral RdRPs, that their constricted active site might serve to increase fidelity, and that their conserved active site motif can be modified to modulate biochemical activities.
Studies of unusual catalysis : a tale of four enzymes from diverse biosynthesis pathways
(2013-12-17) Sun, He, Ph. D.; Liu, Hung-wen, 1952-; Anslyn, Eric V; Fast, Walter L; Kerwin, Sean M; Whitman, Christian P
The diverse reactions that enzymes catalyze have fascinated enzymologists for decades. Continuing investigations in the biosynthesis of both primary and secondary metabolites have led to the discovery of enzymes that employ unusual ways to mediate bio-transformations. Exploration of such atypical biological catalysts is not only important for a comprehensive understanding of the natural products biosynthesis, but also providing new insights that are potentially valuable for developing novel compounds with enhanced biological activities. This dissertation describes the characterization of four enzymes that demonstrate unusual catalytic properties in different biosynthesis pathways. UDP-galactopyranose mutase (UGM) is required for cell wall biosynthesis in many microorganisms. It uses the common cofactor flavin adenine dinucleotide (FAD) in an unusual manner. Positional isotope exchange and kinetic linear free energy relationship studies provide a direct experimental evaluation of the nucleophilic participation by reduced FAD during UGM catalysis. MoeZ from Amycolatopsis orientalis is a unique sulfur carrier protein (SCP) activating enzyme that participates in the metabolism of sulfur. Unlike reported pathway specific homologues, MoeZ can activate multiple SCPs from different biosynthesis pathways found in A. orientalis. Herein, the enzyme is characterized biochemically, and a disulfide intermediate is suggested as part of its catalytic cycle for sulfur transfer from thiosulfate to SCPs. The last two enzymes, Fom3 and OxsB, belong to the cobalamin-dependent radical SAM class of enzymes. While these enzymes are believed to operate with unprecedented chemistry, they remain poorly understood and understudied. Fom3 is proposed to methylate an unactivated carbon center in the biosynthesis of fosfomycin, which is a clinically relevant antibiotic. OxsB is responsible for the biosynthesis of oxetanocin A, an oxetane ring containing nucleoside that exhibits antiviral activity. Efforts have been made to isolate active Fom3 and reconstitute the in vitro activity of OxsB. Reductive cleavage of SAM by the latter enzyme has been demonstrated for the first time and is described in this dissertation.
The Kinetic Mechanism of Multisite Phosphorylation of Activating Transcription Factor 2 by c-Jun N-terminal Kinase 2
(2024) DeJong, Audrey; Dalby, Kevin
The mitogen-activated protein kinases (MAPKs) signal transduction pathway has been well studied as a regulator of a wide range of cellular responses. Specifically, the three-kinase cascade that activates activating transcription factor 2 (ATF2) is implicated in cell proliferation, stress responses, and DNA damage repair. 1 While the kinetic mechanism of ATF2 phosphorylation by p38a has been shown to be non-processive, the phosphorylation mechanism of ATF2 by c-Jun N-terminal kinase 2 (JNK2) is currently unknown. 2 Here, we will investigate the steady-state kinetic mechanism of ATF2 phosphorylation by JNK2 by comparing its kinetic profile with a lower-affinity mutant JNK2 (R127A). Our results show that JNK2 (R127A) has a lower affinity for ATF2 (K m = 4.75 µM ± 0.92) with a similar catalytic efficiency (k cat /K m = 25 s -1 µM -1 ) to the wild-type JNK2 (k cat /K m = 20 s -1 µM -1 ). This data helps design future pre-steady-state kinetic experiments to investigate this mechanism further.
Unveiling the architectures of five bacterial biomolecular machines
(2014-08) Fage, Christopher Dane; Keatinge-Clay, Adrian Tristan; Hoffman, David W; Whitman, Christian P; Appling, Dean R; Iverson, Brent L; Hackert, Marvin L
Natural products represent an incredibly diverse set of chemical structures and activities. Given this fathomless, ever-evolving diversity, a reasonable approach to designing new molecules entails taking a closer look at the biochemistry that Nature has crafted over billions of years on Earth. In particular, much can be learned by unveiling the architectures of proteins, life’s molecular machines, through methods like X-ray crystallography. Acquiring the blueprints of an enzyme brings us closer to understanding the mechanism by which the enzyme transforms a simple substrate it into a complex product with biological function, and inspires us to engineer such systems to our own ends. With a focus on macromolecular structural characterization, this document elaborates on five Gram-negative bacterial biosynthetic enzymes from two categories: Cell-surface modifiers and polyketide synthases. Among the first category are the glycyl carrier protein AlmF and its ligase AlmE of Vibrio cholerae and the phosphoethanolamine transferase EptC of Campylobacter jejuni. These proteins are responsible for decorating cell-surface molecules (e.g., lipid A) of pathogenic bacteria with small functional groups to promote antibiotic resistance, motility, and host colonization. AlmE and EptC represent potential drug targets and their structures lay the groundwork for the design of therapeutics against food-borne illnesses. Included in the second category are the [4+2]-cyclase SpnF and two ketoreductase-linked dimerization elements, each from the spinosyn biosynthetic pathway in Saccharopolyspora spinosa. The former catalyzes a putative Diels-Alder reaction to form a tricyclic precursor of the insecticide spinosad, while the latter two organize ketoreductase domains within modules of a polyketide synthase. The second category also includes Ralstonia eutropha β-ketoacyl thiolase B, a substrate-permissive enzyme that can make or break carbon-carbon bonds with assistance from Coenzyme A or an analogous thiol. Each of these proteins exhibit intriguing structural features or catalyze reactions that show promise for biochemical engineering.