Browsing by Subject "Enzymes"

Now showing 1 - 14 of 14

Biochemical studies of the enzymes involved in deoxysugar D-forosamine biosynthesis
(2004) Hong, Lin, 1976-; Liu, Hung-wen, 1952-
Deoxysugars are indispensable structural components of many biologically active natural products and are essential for many significant cellular processes. In this work, the biosynthetic pathway of TDP-forosamine, which is a 2,3,4,6-tetradeoxy sugar component of the potent, and environmentally benign insecticide spinosyn, has been established without ambiguity. Five genes, spnO, spnN, spnQ, spnR, and spnS have been cloned from Saccharopolyspora spinosa chromosomal DNA and expressed in E. coli. The encoded proteins have been purified and characterized by in vitro assays. The products of four reactions have been isolated and characterized with MS and/or NMR. SpnO was demonstrated to be a 2,3-dehydratase, while SpnN was shown to be a 3- ketoreductase, catalyzing equatorial hydroxyl formation at C-3. Studies of the combined action of SpnO and SpnN reinforce the previous finding that C-2 deoxygenation is accomplished through a -elimination/reduction type mechanism. SpnQ is a homologue of the well-characterized CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1) which in combination with CDP-6-deoxy-4-keto-3,4-glucoseen reductase (E3) catalyzes the C-O bond cleavage at C-3 in the biosynthesis of CDP-ascarylose in Yersinia pseudotuberculosis. In the biosynthesis of TDP-forosamine, SpnQ was shown to catalyze an analogous C-3 deoxygenation but instead using ferredoxin/ferredoxin reductase pair as the electron transfer media. Additionally, in the absence of ferredoxin/ferredoxin reductase to allow C-3 deoxygenation, and in the presence of Lglutamate as an amino donor, SpnQ was shown to act as a 4-aminotransferase, converting TDP-2,6-dideoxy-4-keto-D-glucose to TDP-4-amino-2,4,6-trideoxy-D-glucose. Due to the instability of 2-deoxysugars, an analog of TDP-4-amino-2,3,4,6-tetradeoxy-glucose was used to characterize the late steps in the pathway. SpnR was shown to be a 4- aminotransferase which, interestingly, can recognize both the SpnQ product and the SpnN product as substrates. SpnS was shown to be a 4-amino-N,N-dimethyltransferase. Mechanistic investigation revealed that both N-methyl groups of TDP-forosamine are installed by SpnS in a step-wise manner via a monomethylated intermediate. Together, studies described in this thesis have extensively enriched our knowledge of the enzymes involved in deoxysugar biosynthesis. The newly discovered substrate flexibility of TDPforosamine pathway enzymes will also be very useful in generating engineered sugar biosynthetic pathway for use in generating novel natural products.
The bioelectrochemistry of enzymes and their cofactors at carbon nanotube and nitrogen-doped carbon nanotube electrodes
(2014-05) Goran, Jacob Michael; Stevenson, Keith J.; Crooks, Richard M; Kirisits, Mary J; Keatinge-Clay, Adrian T; Brodbelt, Jennifer S
This dissertation explores the electrochemical behavior of enzymes and their cofactors at carbon nanotube (CNT) and nitrogen-doped carbon nanotube (N-CNT) electrodes. Two common types of oxidoreductases are considered: flavin adenine dinucleotide (FAD)-dependent oxidases and nicotinamide adenine dinucleotide-dependent (NAD⁺)-dehydrogenases. Chapter 1 presents the oxygen reduction reaction (ORR) at N-CNT electrodes as a way to electrochemically measure enzymatic turnover at the electrode surface. The unique peroxide pathway at N-CNT electrodes, which catalytically disproportionates hydrogen peroxide (H₂O₂) back into oxygen, provides an increased ORR current directly proportional to the rate of enzymatic turnover for H₂O₂ producing enzymes, even in an oxygen saturated solution. Biosensing of L-lactate using the increased ORR current is demonstrated using L-lactate oxidase. Chapter 2 explores the surface bound electrochemical signal of FAD when FAD-dependent enzyme or free FAD is allowed to spontaneously adsorb onto the CNT/N-CNT surface. Specifically, the origin of the enzymatically generated FAD signal and the rate constant of the electron transfer are elucidated. Chapter 3 continues the discussion of the cofactor FAD by demonstrating its use as an informative surface specific redox probe for graphitic carbon surfaces. Primarily, FAD can be used to determine the electroactive surface area and the relative hydrophobicity/hydrophilicity of graphitic surfaces. Chapter 4 changes gears to NAD⁺-dependent dehydrogenases by investigating the electrocatalytic oxidation of NADH at N-CNTs in comparison with conventional carbon electrodes or nondoped CNTs. Biosensing of glucose through the oxidation of NADH is demonstrated using glucose dehydrogenase adsorbed onto the N-CNT surface. Chapter 5 continues the discussion of NAD⁺-dependent dehydrogenases by addressing the reaction kinetics of NADH oxidation at N-CNTs as a tool to measure the enzymatic reduction of NAD⁺.
Characterization of the activities of trans-3-chloroacrylic acid dehalogenase and cis-3-chloroacrylic acid dehalogenase and malonate semialdehyde decarboxylase homologues : mechanism and evolutionary implications
(2009-12) Serrano, Hector, doctor of pharmacy; Whitman, Christian P.
Members of the tautomerase superfamily are characterized by a [beta-alpha-beta] structural fold motif as well as a catalytic N-terminal proline (Pro-1). Three members of the superfamily are involved in the degradation of the nematocide 1,3-dichloropopene; trans-3-chloroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD) and malonate semialdehyde decarboxylase (MSAD). CaaD and cis-CaaD are involved in the hydration of their respective 3-chloroacrylic acid isomers to generate malonate semialdehyde. Subsequently, MSAD is responsible for catalyzing the decarboxylation of malonate semialdehyde to generate acetaldehyde. All three of these enzymes contain an N-terminal proline (Pro-1) that functions as a general acid, in contrast to other tautomerase superfamily members, such as 4-oxalocrotonate tautomerase (4-OT) and macrophage migration inhibitory factor (MIF), where Pro-1 acts as a catalytic base. Two new members of the tautomerase superfamily have been cloned and characterized; FG41 MSAD, a homologue of MSAD from Coryneform Bacterium strain FG41, and Cg10062, a homologue of cis-CaaD from Corynebacterium glutamicum, with low-level cis-CaaD and CaaD activities. As part of an effort to delineate the mechanisms of CaaD, cis-CaaD and Cg10062, secondary activities for all three enzymes were characterized. The three enzymes function as efficient phenylpyruvate tautomerases (PPT), converting phenylenolpyruvate to phenylpyruvate. The activity also indicates that the active site of these three enzymes can ketonize enol compounds, thereby providing evidence for the presence of an enediolate intermediate. The characterization of FG41 MSAD uncovered an activity it shares with MSAD. FG41 MSAD catalyzes the hydration of 2-oxo-3-pentynoate, but at a rate that is 50-fold less efficient than that of MSAD (as assessed by kcat/Km values). Mutagenesis studies of FG41 MSAD revealed that a single mutation resulted in a 8-fold increase in the activity. The characterization of Cg10062 and attempts to enhance the low-level cis-CaaD activity demonstrated the need for a bacterial screen that could screen a library of mutants. The resulting bacterial screen could be used to screen other members of the superfamily for dehalogenase activity. An in-depth exploration of the Cg10062 and FG41 MSAD activities may lead to a better understanding of the mechanism of cis-CaaD and MSAD and further delineate the evolutionary pathway for the tautomerase superfamily.
Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans
(Public Library of Science, 2009-11-17) Brown, Stacie A.; Whiteley, Marvin
Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen and the proposed causative agent of localized aggressive periodontitis. A. actinomycetemcomitans is found exclusively in the mammalian oral cavity in the space between the gums and the teeth known as the gingival crevice. Many bacterial species reside in this environment where competition for carbon is high. A. actinomycetemcomitans utilizes a unique carbon resource partitioning system whereby the presence of L-lactate inhibits uptake of glucose, thus allowing preferential catabolism of L-lactate. Although the mechanism for this process is not fully elucidated, we previously demonstrated that high levels of intracellular pyruvate are critical for L-lactate preference. As the first step in L-lactate catabolism is conversion of L-lactate to pyruvate by lactate dehydrogenase, we proposed a model in which the A. actinomycetemcomitans L-lactate dehydrogenase, unlike homologous enzymes, is not feedback inhibited by pyruvate. This lack of feedback inhibition allows intracellular pyruvate to rise to levels sufficient to inhibit glucose uptake in other bacteria. In the present study, the A. actinomycetemcomitans L-lactate dehydrogenase was purified and shown to convert L-lactate, but not D-lactate, to pyruvate with a Km of approximately 150 µM. Inhibition studies reveal that pyruvate is a poor inhibitor of L-lactate dehydrogenase activity, providing mechanistic insight into L-lactate preference in A. actinomycetemcomitans.
Development of enzyme-based sensor arrays
(2001-08) Curey, Theodore Edward; Shear, Jason B.
Measuring multiple components in complex mixtures can present significant analytical challenges. Characterization of such samples typically requires sample “pre-treatment” steps. Separations, for example, commonly are used to isolate analytes spatially before being determined sequentially. This process, however, tends to limit the breadth of measurable analytes, and can suffer from poor selectivity and long analysis times. Detection strategies based on molecular recognition have in some cases improved specificity and shortened analysis times; however, these approaches typically have been designed for the detection and quantification of single components present in complex mixtures. Recently, progress has been made towards the development of sensor arrays, combining the speed and selectivity of multiple sensing methods into a single platform. These technologies, however, have largely focused on narrow groups of analytes such as nucleic acids of varying polymer length. The multiplicity of analytes and variations in analyte properties such as molecular mass, charge or hydrophilicity contribute to the challenges of comprehensive solution phase multi-component analysis. We report here the development of enzyme-based sensors that, in combination with other non-catalytic sensors, form the basis of a solution-phase sensor array. In this strategy, enzymes are immobilized onto porous agarose beads, localized within wells etched into a silicon chip, and coupled to fluorometric and colorimetric reporting schemes to form an optically based site-addressable sensor array. In these studies, we have explored factors that impact the immobilization of enzymes onto solid supports (e.g., pH, incubation time) and the changes to enzyme behavior upon immobilization. We demonstrated the concept of using individual sensors to correct other sensors on the same platform. The linear response of a biosensor is important to analytical measurements and we illustrated the extension of a glucose sensor’s linear response. Finally, we demonstrated rapid multi-component sensing in the determination of monosaccharides, disaccharides, and essential wine components.
High throughput directed enzyme evolution using fluorescence activated cell sorting
(2003-05) Olsen, Mark Jon; Iverson, Brent L.; Georgiou, George
Mechanistic studies of HPP epoxidase and DXP reductoisomerase: applications to biosynthesis and antibiotic development
(2008-05) Munos, Jeffrey Wayne, 1979-; Liu, Hung-wen, 1952-
The focus of this dissertation is the study of two enzymes, DXR and HppE. DXR catalyzes the first committed step in the MEP pathway, which is the pathway most eubacteria, archeabacteria, algae, and the plastids of plants use for the biosynthesis of isoprenoid. Since mammals utilize the mevalonate pathway and isoprenoids are essential for survival, all enzymes in the MEP pathway are excellent antibiotic targets. One antibiotic that has promise in the fight against malaria is the natural product fosmidomycin, whose antibiotic activity is due to its ability to bind and inhibit DXR. With a deeper understanding of DXR's catalyzed reaction, it will be possible to design a more sophisticated and potent antibiotic. To probe the mechanism of DXR, two fluorinated substrate analogues, 3F-DXP and 4F-DXP, and a fluorinated product analogue, FCH₂-MEP were designed and analyzed as possible substrates or inhibitors. To further analyze the mechanism of DXR, a 2° [²H]-KIE study was conducted using the equilibrium perturbation method. The second enzyme this dissertation examines is HppE, which catalyzes the final step in the biosynthesis of the antibiotic, fosfomycin. Fosfomycin is a clinically useful antibiotic for the treatment of limb-threatening diabetic foot infections and urinary tract infections. Chemically speaking, HppE is unique for two reasons. First, HppE's epoxidation differs from Nature's standard method of epoxide formation by alkene oxidation, where the epoxide oxygen is derived from molecular oxygen. For HppE, the epoxide is formed through the dehydrogenation of a secondary alcohol; thus the epoxide oxygen is derived from the substrate. Second, HppE is a unique member of the mononuclear non-heme iron-dependent family of enzymes. HppE differs from all other mononuclear non-heme iron-dependent enzymes by requiring NADH and an external electron mediator for turnover but not requiring [alpha]-KG, pterin, ascorbate, or an internal iron-sulfur cluster. After a study was published on the activity of zinc-reconstituted HppE from Streptomyces wedmorensis, the proposed iron and NADH dependent mechanism of HppE was reevaluated and was reconfirmed. The HppE from Pseudomonas syringae (Ps-HppE) was also purified and was characterized biochemically and spectroscopically. The results of [²H] and [¹⁸O]-KIE studies on Ps-HppE are also reported.
Mechanistic studies of two iron-containing enzymes that catalyze unusual chemical transformations
(2011-08) Chang, Wei-chen, Ph. D.; Liu, Hung-wen, 1952-; Anslyn, Eric V; Fast, Walter L; Kerwin, Sean M; Whitman, Christian P
Enzymes are biological catalysts which trigger chemically inert reaction or accelerate the rates of chemical reactions, oftentimes by many orders of magnitude compared to uncatalyzed reactions. The remarkable catalytic ability afforded by enzymes derives not only from the structure and chemical properties of the enzyme active sites, but sometimes involves redox active metal ions, which allow enzymes to selectively bind to their substrates and to stabilize high energy chemical species along the reaction coordiante. To enhance their catalytic ability, many enzymes have also evolved to require metal ions for activity. Metal ions adapted by enzymes often provide crucial chemical functionality and/or reactivity that are not accessible by the twenty canonical amino acids. Metal ion-containing enzymes serve to greatly broaden the diversity of chemical reactions that can be mediated by enzymes. The work described herein focuses on mechanistic studies of two enzymes that use iron to catalyze two distinctive reactions. In the first part of this work, studies will focus on the (S)-2-hydroxylpropylphosphonate epoxidase (HppE), a mono-nuclear non-heme iron containing enzyme that is an essential enzyme in fosfomycin biosynthesis, and employs an unidentified reduction system for catalysis. In biological systems, mono-nuclear non-heme iron containing enzymes mediate C-H bond activation and further diverse to various outcomes. The HppE catalyzed reaction discovered herein involves oxidative carbon-phosphorous bond migration, raising questions as chemical mechanism(s) can account for such an unusual transformation. The chemical mechanism of HppE will be interrogated with a combination of organic synthesis and biochemical techniques. Our current results suggest that the HppE may first employ a novel mode of non-heme iron containing enzymes catalysis involving hydrogen atom removal and followed by a carbocation-triggered C-P bond migration. In the second part of this dissertation, the focus is to elucidate the mechanism of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH), an enzyme that plays a role in regulating the production of the isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IspH is a [4S-4Fe] enzyme that catalyzes a reductive deoxygenation reaction requiring the addition of two electrons during turnover. Although extensive efforts have been devoted to the study of this transformation, the mechanism of this intriguing reaction remains elusive. Our current data provide experimental evidence indicating the role(s) of iron-sulfur cluster and has mechanistic implication for this unusual reaction. Taken together, our studies of HppE and IspH help to illustrate the catalytic diversity of non-heme iron containing enzymes, and provide mechanistic insights into these atypical reactions.
A Model of a MAPK•Substrate Complex in an Active Conformation: A Computational and Experimental Approach
(Public Library of Science, 2011-04-11) Lee, Sunbae; Warthaka, Mangalika; Yan, Chunli; Kaoud, Tamer S.; Piserchio, Andrea; Ghose, Ranajeet; Ren, Pengyu; Dalby, Kevin N.
The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)2-3-X2-6-ΦA-X-ΦB, where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus 7RK-X2-ΦA-X-ΦB13. This results in a 2-fold increase in kcat/Kmfor the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves kcat/Km by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 ofEts results in a 14-fold decrease in kcat/Km, with little apparent change in kcat. A peptide that binds to the DRS of ERK2 affects Km, but not kcat. Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.
Novel high-throughput screening methods for the engineering of hydrolases
(2011-05) Gebhard, Mark Christopher; Georgiou, George; Alper, Hal; Ellington, Andrew D.; Iverson, Brent L.; Maynard, Jennifer A.
Enzyme engineering relies on changes in the amino acid sequence of an enzyme to give rise to improvements in catalytic activity, substrate specificity, thermostability, and enantioselectivity. However, beneficial amino acid substitutions in proteins are difficult to rationally predict. Large numbers of enzyme variants containing random amino acid substitutions are screened in a high throughput manner to isolate improved enzymes. Identifying improved enzymes from the resulting library of randomized variants is a current challenge in protein engineering. This work focuses on the development of high-throughput screens for a class of enzymes called hydrolases, and in particular, proteases and esterases. In the first part of this work, we have developed an assay for detecting protease activity in the cytoplasm of Escherichia coli by exploiting the SsrA protein degradation pathway and flow cytometry. In this method, a protease-cleavable linker is inserted between a fusion protein consisting of GFP and the SsrA degradation tag. The SsrA-tagged fusion protein is degraded in the cell unless a co-expressed protease cleaves the linker conferring higher cellular fluorescence. The assay can detect specific cleavage of substrates by TEV protease and human caspase-8. To apply the screen for protease engineering, we sought to evolve a TEV protease variant that has altered P1 specificity. However, in screening enzyme libraries, the clones we recovered were found to be false positives in that they did not express protease variants with the requisite specificities. These experiments provided valuable information on physiological and chemical parameters that can be employed to optimize the screen for directed evolution of novel protease activities. In the second part of this work, single bacterial cells, expressing an esterase in the periplasm, were compartmentalized in aqueous droplets of a water-in-oil emulsion also containing a fluorogenic ester substrate. The primary water-in-oil emulsion was then re-emulsified to form a water-in-oil-in-water double emulsion which was capable of being analyzed and sorted by flow cytometry. This method was used to enrich cells expressing an esterase with activity towards fluorescein dibutyrate from an excess of cells expressing an esterase with no activity. A 50-fold enrichment was achieved in one round of sorting, demonstrating the potential of this method for use as a high-throughput screen for esterase activity. This method is suitable for engineering esterases with novel catalytic specificities or higher stabilit
On the reactions of trans-3-chloroacrylic acid dehalogenase and a cis-3-chloroacrylic acid dehalogenase homologue, Cg10062 : mechanistic and evolutionary implications
(2015-05) Huddleston, Jamison Parker; Whitman, Christian P.; Johnson, Kenneth A; Kerwin, Sean M; Fast, Walter L; Hoffman, David W
The tautomerase superfamily (TSF) provides an excellent model system to study enzyme specificity, catalysis, and divergent evolution. trans-3-Cholroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), and malonate semialdehyde decarboxylase (MSAD) are three TSF members that catalyze the final reactions in the degradation of the nematocide, 1,3-dichloropropene. All three enzymes have the TSF characteristic beta-alpha-beta fold and catalytic amino terminal proline (Pro-1). Both CaaD and cis-CaaD dehalogenate their respective isomers of 3-chloroacrylic acid yielding malonate semialdehyde. Subsequently, MSAD decarboxylates malonate semialdhyde resulting in acetaldehyde and CO2. Their catalytic and substrate specificities are exquisite considering they share three key and positionally conserved residues. As part of an effort to understand how such specificity evolved, a pre-steady-state kinetic analysis of CaaD was carried out. Alongside a similar study on cis-CaaD, a fluorescent mutant of CaaD was constructed that had minimal kinetic differences from the wild-type. The mutant was validated as an accurate fluorescent reporter of change in enzyme state that allowed for the reaction to be followed using stopped-flow methods. Stopped-flow fluorescence, rapid chemical quench data and ultraviolet spectroscopy were globally fit by computational simulation. The fit resulted in a kinetic mechanism for CaaD affording detailed information about the reaction, including measuring the rate of product release, the rate of chemistry, a previously unknown partially rate-limiting step associated with a conformational change, and the definition of binding constants for both products (MSA and Br-). In addition to the dehalogenation reaction, the reaction of the fluorescent mutant with a mechanism-based inhibitor, 3-bromopropiolate, was characterized. The values for the apparent rate of inhibition and potency were defined and estimates were determined for the values of the rate of chemistry and the release of bromide. The information gathered during these inhibition experiments was used to further refine the CaaD dehalogenation mechanism eliminating ambiguities present in the initial data set. Finally, the reactions of a cis-CaaD homologue, Cg10062 from Corynebacterium glutamicum were characterized. Cg10062 shares high sequence similarity (53%) and the same six critical active site residues as cis-CaaD, but Cg10062 has poor cis-CaaD activity. Moreover, Cg10062 dehalogenates both 3-chloroacrylic acid isomers. The reactions of Cg10062 with propiolate, 2-butynoate, and 2,3 butadienoate were investigated. Cg10062 functions as a hydratase/decarboxylase using propiolate generating malonate semialdehyde and acetaldehyde. Cg10062 catalyzes a hydration-dependent decarboxylation of propiolate as exogenously added malonate semialdehyde is not decarboxylated. With 2,3 butadienoate and 2-butynoate, Cg10062 functions as a hydratase and yields only acetoacetate. Mutations to the activating residues Glu114 and Tyr103 produced a range of results from a reduction in wild-type activity to a switch of activity. Possible intermediates for the hydration and decarboxylation products can be trapped as covalent adducts to Pro-1 when NaCNBH3 is incubated with certain combinations of substrate and mutant enzymes. Three mechanisms are presented to explain these findings along with the strengths and weaknesses of each mechanism in terms of being able to account for experimental observations.
Protein-Protein Docking with F2Dock 2.0 and GB-Rerank
(Public Library of Science, 2013-03-06) Chowdhury, Rezaul; Rasheed, Muhibur; Keidel, Donald; Moussalem, Maysam; Olson, Arthur; Sanner, Michel; Bajaj, Chandrajit
Motivation -- Computational simulation of protein-protein docking can expedite the process of molecular modeling and drug discovery. This paper reports on our new F2 Dock protocol which improves the state of the art in initial stage rigid body exhaustive docking search, scoring and ranking by introducing improvements in the shape-complementarity and electrostatics affinity functions, a new knowledge-based interface propensity term with FFT formulation, a set of novel knowledge-based filters and finally a solvation energy (GBSA) based reranking technique. Our algorithms are based on highly efficient data structures including the dynamic packing grids and octrees which significantly speed up the computations and also provide guaranteed bounds on approximation error. Results -- The improved affinity functions show superior performance compared to their traditional counterparts in finding correct docking poses at higher ranks. We found that the new filters and the GBSA based reranking individually and in combination significantly improve the accuracy of docking predictions with only minor increase in computation time. We compared F2 Dock 2.0 with ZDock 3.0.2 and found improvements over it, specifically among 176 complexes in ZLab Benchmark 4.0, F2 Dock 2.0 finds a near-native solution as the top prediction for 22 complexes; where ZDock 3.0.2 does so for 13 complexes. F2 Dock 2.0 finds a near-native solution within the top 1000 predictions for 106 complexes as opposed to 104 complexes for ZDock 3.0.2. However, there are 17 and 15 complexes where F2 Dock 2.0 finds a solution but ZDock 3.0.2 does not and vice versa; which indicates that the two docking protocols can also complement each other. Availability -- The docking protocol has been implemented as a server with a graphical client (TexMol) which allows the user to manage multiple docking jobs, and visualize the docked poses and interfaces. Both the server and client are available for download. Server: http://www.cs.utexas.edu/~bajaj/cvc/software/f2dock.shtml. Client: http://www.cs.utexas.edu/~bajaj/cvc/software/f2dockclient.shtml.
Sequence, structure, and function relationships in the aldolase and tautomerase superfamilies
(2022-05-06) Lancaster, Emily Beatrice; Whitman, Christian P.; Fast, Walter; Dalby, Kevin; Hoffman, David
Studying the structure of an enzyme and how it relates to its function has been a goal of enzymologists for decades. Although informative and useful, classical techniques, such as BLAST searches or manual analysis of multisequence alignments may be too focused. These techniques tend to rely on comparing a manageable quantity of sequences. As a result, patterns in conservation of certain residues across a limited number of sequences carry more weight in the study. In the first section, we present a study that used these classical approaches to compare only 3 sequences of Aldolase Superfamily members as described below. NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (where the ortho-substituent is H, OH, or CO₂⁻) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is part of a bacterial pathway for the degradation of naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. NahE crystal structures show these core active site elements along with other nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on binding, catalysis, and/or the reaction step (hydration vs aldol cleavage). The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. The Y155F mutation also significantly hinders catalysis and falls in between these extremes. These observations are put into a structural context. Finally, trapping experiments were carried out with substrate, NaCNBH₃, and wild type and selected mutations. The mass spectral analysis is consistent with the observed activities and suggests that pyruvate is released quickly from the active site, but salicylaldehyde is not. In the second section, we utilize a more modern technique of sequence analysis. We present studies done to analyze trends observed within the Tautomerase Superfamily (TSF) that have been identified by a sequence similarity network (SSN). Hidden trends in sequences that appear insignificant at the small scale may be revealed on a larger scale, such as in the second section. The amino-terminal proline (Pro1) has long been thought to be a mechanistic imperative for tautomerase superfamily (TSF) enzymes, functioning as a general base or acid in all characterized reactions. However, a global examination of more than 11,000 nonredundant sequences of the TSF uncovered 346 sequences that lack Pro1. The majority (~85%) are found in the malonate semialdehyde decarboxylase (MSAD) subgroup where most of the 294 sequences form a separate cluster. Four sequences within this cluster retain Pro1. Because these four sequences might provide clues to assist in the identification and characterization of activities of nearby sequences without Pro1, they were examined by kinetic, inhibition, and crystallographic studies. The most promising of the four (from Calothrix sp. PCC 6303 designated 437) exhibited decarboxylase and tautomerase activities and was covalently modified at Pro1 by 3-bromopropiolate. A crystal structure was obtained for the apo enzyme (2.35 Å resolution). The formation of a 3-oxopropanoate adduct with Pro1 provides clues to build a molecular model for the bound ligand. The modeled ligand extends into a region that allows interactions with three residues (Lys37, Arg56, Glu98), suggesting that these residues could play roles in the observed decarboxylation and tautomerization activities. Moreover, these same residues are conserved in 16 nearby, non-Pro1 sequences in a sequence similarity network. Thus far, these residues have not been implicated in the mechanisms of any other TSF members. The collected observations provide starting points for the characterization of the non-Pro1 sequences. Five non-Pro1 sequences were studied in this section, as well, containing either glycine, alanine, valine, threonine, or serine at the N-terminus. The most promising was NJ7 (from Nostoc sp. strain PCC 7120/SAG 25.82/UTEX 2576 derived from the UniProt Accession code Q8YNJ7). Kinetic analysis showed that this enzyme with Val1 has tautomerization and decarboxylation activity. The introduction of Pro1 enhanced NJ7’s performance as a tautomerase and decarboxylase.
Transiently Transfected Purine Biosynthetic Enzymes Form Stress Bodies
(Public Library of Science, 2013-02-06) Zhao, Alice; Tsechansky, Mark; Swaminathan, Jagannath; Cook, Lindsey; Ellington, Andrew D.; Marcotte, Edward M.
It has been hypothesized that components of enzymatic pathways might organize into intracellular assemblies to improve their catalytic efficiency or lead to coordinate regulation. Accordingly, de novo purine biosynthesis enzymes may form a purinosome in the absence of purines, and a punctate intracellular body has been identified as the purinosome. We investigated the mechanism by which human de novo purine biosynthetic enzymes might be organized into purinosomes, especially under differing cellular conditions. Irregardless of the activity of bodies formed by endogenous enzymes, we demonstrate that intracellular bodies formed by transiently transfected, fluorescently tagged human purine biosynthesis proteins are best explained as protein aggregation.