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Site Directed Mutagenesis and Study of Oxalate Decarboxylase and its Isozyme Lucas A. Cathey Undergraduate Honors Thesis Spring 2023 Department of Chemistry, University of Florida
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2 CONTENTS 3 4 9 Site 9 .. 10 4 8 8 Structure of N163F and OxDC WT 9 Activity of N163F and OxDC WT 3 Radical Signatures for N163F and OxDC WT 6 Conclusion 9 2 9 Acknowledgments 3 1 References 3 2
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3 ABSTRACT Oxalate decarboxylase (OxDC) is a naturally occurring enzyme which catalyzes the decarboxylat ion of the mono anion of oxalic acid in acidic conditions , a mechanism which has potential applications in both industrial and medical fields if it can be optimized and understood. 1, 6 The enzyme also possesses a naturally occurring isozyme, OxDD, which performs identical function with half the activity in natural conditions, and by determining the source of this difference a deeper understanding of the function of OxDC and its relation ship with this isozyme can be obtained. 7 The structures of the two enzymes possess a major discrepancy in a flexible peptide loop near the active site which has been demonstrated to effect enzymatic activity, so through site directed mutagenesis the pepti de loop of OxDC was altered to resemble that of OxDD; an asparagine in the loop was replaced with phenylalanine, a larger and less polar amino acid . 9 Though it was expected that this alteratio n would result in a reduction in activity for the mutant as compared to the wild type ( WT ) of OxDC, testing has demonstrated that the inhibition of the peptide loop in this manner actually produces negligible changes in the overall functi on of the enzyme , though it does produce some structural differences . Given these results, it can be concluded that some other difference between the two isozymes is responsible for the greater activity of OxDC.
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4 INTRODUCTION Oxalate decarboxylase (Ox DC) is an enzyme which catalyzes the decarboxylation of the mono anion of oxalic acid and is induced in acidic conditions (optimal activity is in the range of pH 4 5) , producing carbon dioxide and a formate ion, as shown in Scheme 1. 1 Scheme 1 The reaction catalyzed by OxDC. Though not consumed by the reaction, oxygen is necessary for catalytic activity. Figure created by the author. The enzyme utilizes cofactors of dioxygen and manganese, with the metal incorporating into the enzyme at a ratio of two atoms per monomer in the structure , and naturally occurs in the bacterium Bacillus subtilis . The monomer itself is a pair of beta barrel structures, with the manganese atoms distribut ed into C terminal and N terminal positions. The tertia ry structure of OxDC is that of a hexamer, with three monomers forming a triangular trimer structure, and two of the trimers layering on top of one another , as shown in Figure 1 . 2
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5 Figure 1 The crystal structure of OxDC with Ma n ganese highligh ted in purple and pink. A) Monomeric structure. B) Hexameric structure. 2 Figures constructed by the author using PyMOL . 3 In each monomer, the active site is located on the N terminal manganese , which coordinates to the oxalic acid mono anion during catalysis, which occurs under acidic stress. The currently proposed mechanism for this catalysis is shown in Scheme 2 . 4 A) B)
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6 Scheme 2 The current proposed mechanism for OxDC. 4 Figure created by the author.
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7 By optimizing this catalysis, the eventual hope is that OxDC might be used in the industrial sector for the decomposition of oxalate or as a therapeutic agent for kidney stone, of which calcium oxalate are a large component. 6 In order to advance the understanding of OxDC and its mechanism, these experiments study it in tandem with one of its naturally o ccurring isozymes, designated OxDD. OxDD performs an identical function to OxDC in natural conditions, but with approximately half the decarboxylase activity and with an additional oxidase activity. 7 The reason for this difference is currently unknown, which prompted some investigation into the amino acid sequences of both isozymes in order to determine differences within their structures that might contribute to it. Utilizing the National Center for Biotechnology Information (NCBI) NCBI Blast tool , the peptide sequences were compared, leading to the discovery of a relevant discrepancy in a flexible peptide loop . 8 The loop , shown in Figure 2, consists of five residues , S 161 E 162 N 163 S 164 T 165 , in OxDC; and in OxDD it is identical with the exception of the asparagine being replaced by a phenylalanine : S 169 E 170 F 1 71 S 172 T 173 .
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8 Figure 2 The flexible peptide loop near the active site, with the residues highlighted to differentiate them from the surrounding structure. 2 Figure constructed by the author using PyMOL. 3 This flexible loop has been proven to be involved with gating access to the active site, allowing substrate access to the N terminal Manganese and products to leave . 7 Additionally, its alteration to become more open causes radical leakage from the active site and reduces activity. 9 The phenylalanine, far bulkier a nd less polar than asparagine, may inhibit the ability of OxDD to hold onto intermediates and control access to the active si te. In order to test this hypothesis, OxDC was manipulated through site directed mutagenesis in order to determine the effect of the active loop on catalysis. The wild type ( WT ) of OxDC was tested against a mutant in which the active loop of the enzyme was altered to be identical to that of OxDD, designated N163F. By determining the activity and structure of both species, it can be determined if the difference in the structure of the active loop leads to any significant conformational differences in the enz yme or is the root cause of the difference in activity for OxDC and OxDD.
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9 MATERIALS AND METHODS Site Directed Mutagenesis Mutation was achieved through the usage of non overlap polymerase chain reaction (PCR), using primers which were manufactured using a Q5 Site Directed Mutagenesis Kit from New England Biolabs and designed using New England BioLabs NE BaseChanger to incorporate the mutation , with the resulting primers shown in Table 1 , with the mutation highlighted . Table 1 Site Directed Mutagenesis Prim ers Mutant Primer N163F Forward ATTCTCTGAA ttt AGCACGTTCCAGCTGA Reverse GATCCATCGTCAAACACGAGCA PCR and mixed , free water 32 vector . This mixture was then subjected to 25 cycles of PCR under the following conditions: a 30 secon d initial denaturation step at 98ºC, a further 10 second denaturation step at the same temperature, a 30 second annealing step at 58ºC, a 3 minute and 18 second extension step at 72ºC, a final extension for 3 minutes at the same temperature, and lastly a h olding step for 10 minutes at 4ºC. Once the reaction was complete, the products were tested for a successful reaction using an agarose gel, manufactured using 0.4 g agarose, 4 mL of TAE Buffer (0.4 M tris(hydroxymethyl)aminomethane (Tris) acetate, 0.01 M e thylenediaminetetraacetic acid [EDTA], pH 8.3) , 36 mL of de ethidium bromide, added to the solution after it was brought to a boil and then cooled to just
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10 below boiling. Visual analysis of the PCR products using ultraviolet ligh t confirmed a successful reaction for the N163F mutant . Induction and Purification of Enzymes In order to test for the success of the mutation, the PCR products were introduced into the nuclease deficient Escherichia coli ( E. coli optimize the recovery of intact DNA from the sample. The PCR products were prepared for inoculation using KLD, at a New England Biolabs (NEB) KLD mix ( a combination of kinases, ligases, and Dpnl enzymes 1 .5 free water NEB KLD buffer equilibrate on ice, and heat shocked at 42ºC for 30 seconds in order to encourage inoculation. of Luria Broth (LB) Media (5 g yeast extract, 10 g tryptone, 5 g sodium chloride, 1 L nuclease free water), the E. Coli was left to propagate for 1 hour before a chlo ride, 2.5 g yeast extract, 5 g tryptone, 7.5 g agar, 500 mL nuclease free water; autoclaved develop on the agar plates for approximately 15 hours at 3 7 ºC, and upon visual confirmation of successful growth, two isolated colonies were harvest ed using a glass rod and introduced into triplicate . These cultures were then allowed to grow for an additional 15 hours at 3 7 ºC an d shaking at 200 rpm, and then used for DNA isolation. Isolation of the DNA was accomplished first through use of the Wizard Plus Miniprep kit, or as follows: first was performed a centrifugation at 11641 rpm for 5 minutes of the overnight cultures, in ord er to concentrate and isolate the cell matter. These concentrated cell
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11 resuspension solution , and the cells were lysed with cell lysis solution rotease solution was introduced to the mixture for 5 minutes, before the lysate reaction was neutralized neutralization solution . With the cells lysed, the freed DNA was isolated through centrifugation at 1 3774 rpm for 15 minutes, which isolated the DNA in solution and reduced cell waste to a pellet. The supernatant was then cleaned using a silica column which traps DNA, which was washed first with the solution and then with two rounds of column wash solution (750 3774 rpm ). The DNA was then free water, and centrifugation for 1 minute at 1 3774 rpm . Successful isolation of the DNA was confirmed through use of a microvolume spectrometer , and the success of N163F mutation appearing in the E. Coli DNA was confirmed via Sanger Sequencing. Successful mutation was then followed by mass production of the mutated enzyme as well as the OxDC WT . This was performed through introduction of the isolated DNA of the N163F mutant and of OxDC WT to E. Coli , though this time to the protease deficient BL21DE3 strain in order to assist in maximum recovery of the OxDC strains. The DNA was introduced into the E. Coli through much the s plates which were allowed to grow at 37ºC for approximately 15 hours. From these plates, two to three colonie s were used to prepare an overnight culture of 50 mL of LB media and 50 mL of nuclease culture was introduced in two portions of 5 mL and two portions of 7 mL into fo ur larger LB
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12 inoculated, these four mixtures were allowed to incubate at 37ºC and 200 rpm until the optical density of the E. Coli had reached a value of 0.5 as d etermined through ultraviolet/visible light spectroscopy measurements taken at 600 nm. Once this value was confirmed, the mixtures were heat shocked at 42ºC for 15 minutes with periodic mixing, and then inoculated with 4.6 mL of 1 M manganese chloride and D 1 thiogalactopyranoside (IPTG). The purpose of the manganese chloride was to provide the necessary manganese content for OxDC to function, while the the IPTG acted as an agent to prevent any inhibition of the production of OxDC. The pET 32 vector for E. Coli works through t he usage of the lac operon, which in natural conditions is eventually inhibited through the presence of a repressor, ending the production of enzyme. This repressor can be removed from the lac operon using lact ose, which is gradually depleted by cell processes, and IPTG acts as a lactose equivalent which is not processed by E. Coli , allowing for unlimited gene expression and the overproduction of OxDC. 10 The four LB Media mixtures, after being induced to overe xpression by IPTG, were left to react for 4 hours at 37ºC and 200 rpm. After this, they were each centrifuged at 6000 rpm for 18 minutes to isolate the cells for lysing and recovery of the produced OxDC. This recovery was performed through affinity chromat ography, using a histidine 6 tag which was encoded into the vector used for the mutation of N163F and the production of the wild type OxDC. With the supernatant disposed of, the cell pellets were lysed using a prepared lysis buffer (1.5 g Tris, 7.30 with nuclease free water and pH 7.5) and sonification. Th e sonification was performed at an amplitude of 70 for 20 cycles, each cycle lasting 30 seconds with a 130 second rest period in between cycles. The lysate was then centrifuged at 11000 rpm for 20 minutes at 4ºC to isolate the freed protein from the cell w aste. The supernatant from this centrifugation was then
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13 introduced to 5 mL of cooled and suspended nickel NTA resin, which had been washed with 8 portions of 5 mL of a prepared wash buffer (1.5 g Tris, 7.30 g sodium chloride, 0.3404 g imidazole, total volu me 250 mL with nuclease free water and pH 8.5), and left to mix on ice for 2 hours to allow the enzyme to attach to the resin via the histidine 6 tag. After this mixing period, the resin was re concentrated via centrifugation at 4500 rpm for 10 mi nutes at 4ºC . The concentrated resin was then introduced to a column and manually washed with the prepared wash buffer with 8 portions of 5 mL , keeping all solutions sufficiently chilled by performing the process in a cold room kept at 20ºC . The enzyme was then eluted from the column using 6 portions of 5 mL of a prepared elution buffer (1.5 g Tris, 7.30 g sodium chloride, 4.25 g imidazole, total volume 250 mL with nuclease free water and pH 8.5). This eluate was then transferred to a buffer more con genial to long term storage of the enzyme (12.114 g Tris, 58.58 g sodium chloride, 2 L total volume with nuclease free water and pH 8.5) , as the imidazole used to displace the enzyme from the Ni NTA column also tends to strip it of its manganese content . 9 This transfer was accomplished through dialysis for 2 hours in 2 L of the storage buffer, a process which was repeated in triplicate to ensure full transfer. Upon completion of the dialysis, the protein solutions were mixed with 1g of BT Chelex 100 resin p er 10 mL of solution, in order to chelate and bind any excess metals in the solution typically this consists of leftover nickel from the column, which is chelated by the resin and can be then cleansed from the solution via centrifugation and elution in a simple column where the Chelex serves as the stationary phase. After mixing for an hour on ice, the centrifugation was performed at 4500 rpm for 10 minutes at 4ºC, along with the elution. The eluate was then concentrated using 30,000 micron filters in the centrifuge at 4500 rpm and 4ºC for 4 minute cycles, continually replenishing the contents of the filters until all of the eluate had been
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14 From here, a Bradford Assay was performed to verify the concentra tion of the protein, using the series listed in Table 2. Once reacted for 20 minutes in the absence of light, a curve was obtained by measuring absorbance at 595 nm and using a standard bovine serum curve for normalization. 12 Table 2 Solutions used for Bradford Assay Trial 1 2 3 4 980 980 980 980 20 19 18 19 0 1 0 0 0 0 2 1 Finally, successful comparison of the conformation of the N163F mutant and OxDC WT was achieved via Native Polyacrylamid e Gel Electrophoresis ( PAGE ) , which was performed using a mini PROTEAN Bio Rad gel and a tris/glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3) , to which 5 mi , diluted to 10 mg/mL, were added in each lane . Current was run through the solution at 150 V and 250 mA for an hour to obtain the results , which were visibly analyzed once the gel had been treated with a heated staining solution (metha nol, acetic acid, Coomassie blue dye). Testing A variety of tests were performed on the obtained OxDC WT and N163F protein strains, the foremost being a measure of activity obtained through an end point assay using formate
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15 dehydrogenase (FDH) . 1 3 As OxDC does not create products easily detected via spectroscopy, FDH is used to react with the produced formate to convert nicotinamide adenine dinucleotide (NAD+) to its protonated form (NADH) , as shown in Scheme 3 , which is easily detected at 340 nm u sing a UV/Vis spectrometer. 1 4 Scheme 3 The reaction catalyzed by FDH. Figure constructed by author. The amount of NADH produced by FDH correlates directly to the amount of formate produced by OxDC, allowing for creation of an activity curve and the ap plication of Michaelis to 5 mg/mL, to a series of increasingly dilute oxalate samples, as shown in Table 3.
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16 Table 3 Oxalate and Formate concentration used in the FDH Assay Oxalate Sample Concentrations (mM) Formate Standard Concentration (mM) 0 0 1.92 0.025 2.88 0.05 3.8 5 0.1 15. 4 0.2 48. 1 0.3 96. 2 0.4 0.3 g Tris, 0.5231 g 2 [Bis(2 hydroxyethyl)amino] 2 (hydroxymethyl)propane 1,3 diol [Bis tris] , 0.2154 g piperazine, 0.52535 citric acid, 50 mL nuclease free w X surfactant , and phenylenediamine (o PDA) . The complete mixture was heated to 25ºC, and upon temperature equilibration the diluted enzyme was added and allowed to react for 1 minute, followed by a que free water, 2 mL of 15 mM phosphate buffer for a pproximately 13 hours at 37ºC. This process was completed in triplicate and the absorbance values for the samples were compared to a set of formate standards prepared similarly, as shown
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17 in Table 3. As the formate standards were not reacted with any OxDC, the contents of the master X surfactant and 10 PDA) in order to accurately refl ect the contents of the samples, but aside from these two differences the formate standards were subjected to the same procedure as the oxalate samples. In order to normalize the obtained Michaelis Menten kinetics data from the FDH assay per metal content of the proteins, a metals assay was performed using inductively coupled plasma atomic spectroscopy , with samples prepared int a metal free version of the previously mentioned storage buffer. Additionally, in order to ascertain if the mutation had any signi ficant effect on the function of the active site, electron paramagnetic resonance (EPR) tests were performed using 3,4 dihydro 2 methyl 1,1 dimethylethyl ester 2H pyrrole 2 carboxylic acid 1 oxide (BMPO) spin trapping. The EPR protein samples (at 10 mg/mL) diethylenetriaminepentaacetic acid In this mixture the most relevant ingredient is the BMP O, which serves as a spin trap for radicals in the reaction solution that allows them to be detected via EPR. 1 5 After being allowed to react for about 2 minutes, the samples were tested at 30ºC with a field sweep of 312 to 362 mT . More structural data was obtained by circular dichroism (CD) spectroscopy to identify any differences in the broader conformation of the mutant as compared to the wild type. These samples were prepped via dialysis of the protein into a 50 mM phosphate buffer at pH 7 with fluoride for analysis by the spectrometer.
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18 RESULTS AND DISCUSSION Mutagenesis of N163F The success of the PCR reaction was confirmed through the use of an agarose gel, shown in Figure 3. Figur e 3 The agarose gel performed on the PCR products for His tagged OxDC WT and the N163F mutant, with the successful results highlighted with red arrows. As seen in the gel, kb DNA ladder in the leftmost lane of the gel, which is the correct weight lane for the desired PCR vectors. Multiple annealing temperatures were attempted for the PCR reaction, and the high lighted lanes represent the most successful temperatures as they fluoresce the most under UV light. Sanger sequencing later confirmed that the mutations were successfully integrated into the vectors, and allowed for the overexpression of the wild type and N163F mutant using the BL21DE3 strain of E. Coli .
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19 Structure of N163F and OxDC WT In order to determine whether the mutation to the flexible loop resulted in any conformational difference between the N163F mutant and the wild type , a Native PAGE was run, with the results showcased in Figure 4. Figure 4 The Native PAGE performed on OxDC WT and the N163F mutant, with labeled lanes and significant bands. The PAGE revealed that, much like the wild type , the N163F mutant organizes itself as a hexamer . However, there was also significant concentration of the mutant protein at the weight corresponding to the monomeric conformation of OxDC, indicating that the mutation makes it more difficult for OxDC monomers to pair up in their trimer configuration , much less the ideal WT N163F Hexamer Trimer Monomer Dimer
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20 hexameric form . However, this could also simply be an artefact of the PAGE, given that the OxDC WT lane, which should have a predominant hexameric band a little else, seems to show significant population of dimers and monomers, as well as f ragments lower on the PAGE, In order to confirm if the N163F mutant might actually have trouble organizes itself as a hexamer, CD spectroscopy was performed to gain insight into the secondary structure and if it differs significantly from the wild type. The spectra obtained via experimentation were processed and interpreted using the E ötvös Loránd University Be ta Structure Selection (BeStSel) software, which produced the plots fo r OxDC WT and N163F shown in Figure s 5 and 6 . 1 6
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21 Figure 5 The fitted CD spectra for OxDC WT, as computed using BeStSel software. The percentage makeup of the enzymes is shown in a pie chart above the spectra, and shows that OxDC is largely made up of beta sheets : Anti1, 2 and 3 represent beta structures were strands are positioned anti parallel to each other, while Parall el represents parallel beta sheets
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22 Figure 6 The fitted CD spectra for the N163F mutant , as computed using BeStSel software. The percentage makeup of the enzymes is shown in a pie chart above the spectra, and shows that the mutant possess a signif icantly higher alpha helix (Helix1 and Helix2) composition than the wild type. The CD spectra reveal more about the internal structure of the mutant and wild type than a Native PAGE, and demonstrate that the secondary structure of the N163F mutant actually bears some large discrepancies from the conformation of the wild type. It appears the mutation has impaired the ability of OxDC to organize with the same secondary structure of the wild type, and
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23 resulted in a larger percentage of the structure being composed of alpha helices than is typical for OxDC, which largely consists of beta sheet structures with very few helices. There is signif icant noise in the segment of the spectrum (about 190 200 n m) which corresponds to the helices, so it is possible the percentage composition of them for N163F is not so high as 41.8%, but the following beta section after 200 nm also is missing some key fea tures when compared to the spectrum f or the wild type , assuming a sharper an d larger shape that indicates the beta structure composition is less in N163F than in wild type. This structural difference could explain the large amount of monomer for N163F in t he Native PAGE, as the disrupted secondary structure of the enzyme could inhibit it from properly forming its trimeric and hexameric superstructures. Activity of N163F and OxDC WT Activity measurements for OxDC WT and N163F were obtained through the FDH A ssay, with triplicate runs providing an aggregate set of kinetics data. This data was summarily modeled using standard Michaelis Menten Kinetics, as shown in Scheme 4 , returning parameters for the catalytic rate constant ( k cat ) and the Michaelis constant ( K M ) . Scheme 4 The form of Michaelis V max (limiting rate), [A] (concentration of substrate), and K M (Michaelis constant). 1 7 Additionally, as adequate manganese incorporation is necessary for full activity of OxDC, Manganese content figures were obtained using inductively coupled plasma mass spectroscopy (ICP MS) so that the determined catalytic efficiency ( k cat /K M ) could be normalized for manganese incorporation, allowing for comparison between the mutant and the wild type .
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24 The full results are shown in Table 4 , with calculated N163F values compared to known values for OxDC WT . A full Michaelis Menten plot for N163F is shown in Figure 7 . Table 4 Accumulated Kinetics for OxDC , OxDD, and various Mutants OxDC WT 1 8 N163F OxDD WT 1 9 F171N 1 9 F171Y 1 9 Mn/monomer 1.93 1.41 1.94 1.10 0.74 K M (mM) 33.3±0.4 32.5±0.7 25.4±2.7 30.6±2.1 14.3±0.73 k cat (s 1 ) 89.2±1.4 83.7±5.5 14.0±0.73 33.3±0.61 5.25±0.35 k cat /Mn (s 1 ) 46.2±0.7 66.3±3.9 7.16±0.20 30.2±0.56 7.10±0.48 k cat /Mn/K M (s 1 mM 1 ) 1.39±0. 0 27 1.83±0.32 0.29±0.032 0.99±0.070 0.50±0.042
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25 Figure 7 Michaelis Menten plot for N163F, with the averaged trial data displayed as points and the average calculated Michaelis Menten curve shown in black. Error bars are shown for the last three data points for the first four, standard deviation was less tha n 1 between the trials. The kinetics data shows that, contrary to expectation, N163F does not exhibit less activity than the wild type . T hough the catalytic efficiency is somewhat higher, the error bounds on the data overlap with those of the wild type, so it cannot be said to be a significant difference . The Michaelis constant, as well, is comparable to that of wild type despite the lower manganese content of the mutant. Comparing this to parallel data taken on OxDD WT and mutations of its active loop (F17 1N altering it to resemble that of OxDC, and F171Y altering it to a theoretically similar tyrosine), it is clear that while the reverse mutation has a significant effect on the activity of OxDD, the same cannot be said when discussing N163F and OxDC. The F171N mutation shows that the replacement of Phe 171 with an asparagine in OxDD improves the activity of the
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26 enzyme, but when the reverse is performed upon OxDD there is negligible effect . 1 9 This suggests that some additional factor is at play in the discrepancy of act ivity between OxDC and OxDD, which allows OxDC to compensate for the impairment of its peptide loop and altered conformational structure. Radical Signatures for N163F and OxDC WT To get a clearer picture of whether the alteration of the active loop in OxDC affects the ability of the substrate to bind in any way, or if any radical leakage might result, the EPR measurements can be consulted. Using parameters for superoxide and carbon dioxide radicals, the EPR data for both OxDC WT and N163F were fit to the pa rameters as shown in Figure 8 .
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27 Figure 8 The EPR data for both OxDC WT and N163F, fit to the known parameters for superoxide and carbon dioxide radical signatures. The raw data is shown in blue, with the fit in orange.
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28 The EPR data confirms what the kinetics data hinted at: the alteration of the peptide loop in OxDC has no e ffect on the ability of the enzyme to hold onto intermediates, as the N163F mutant produces almost the exact same signature as that of OxDC WT . This signature indicates a large percentage of the superoxide radical in the solution, with little carbon dioxid e radical, which would be produced by the leakage of intermediate from the active site, to complement it. A look at the hyperfine coupling constants for the superoxide and carbon dioxide radicals, shown in Table 5 , confirms this. Table 5 Hyperfine Co upling Constants for OxDC WT and N163F Enzyme OxDC WT N163F Radical OOH (1) OOH (2) CO 2 OOH (2) OOH (1) CO 2 g factor 2.0053 7 2.005 51 2.00 199 2.0054 3 2.0055 2 2.001 55 1 H Parameter (mHz) 34. 8 28.8 4 8.0 3 4.8 28. 4 4 7.8 14 N Parameter (mHz) 3 8.3 36.6 39. 6 37. 7 36. 7 40. 4 Weight (%) 79.5 20.1 0.4 87.7 12.3 0 The hyperfine coupling constants are largely consistent between the two enzymes, with no parameter between them differing by more than 1.0 mHz. The two parameter sets for the superoxide radical represent two different conformers that can exist when BMPO bi nds to the superoxide radical at pH 4.0, an O 2 radical and an HO 2 radical. 20 Equilibrium favors the hydroperoxyl radical (Conformer 1), which is the reason for the difference in weights between the two parameter sets, which in any case should not be taken too literally given the equilibrium
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29 relationship between the two conformers. 2 1 ,2 2 However, the discrepancy between the weights of the superoxide parameters and the carbon dioxide radical parameters is significant, and demonstrates both the wild type and th e N163F mutant produced predominantly superoxide radicals, with negligible amounts of carbon dioxide radical showing up in solution. Since it has been proven that only carbon dioxide radical leaks from the active site when the active loop is altered or impaired, it is certain that the N163F mutation has a ne gligible effect on the radical activity of the enzyme. 20 CONCLUSION Though the flexible peptide loop has been proven to demonstrate an effect on the catalysis of OxDC, it has become clear that the alteration of this loop in the isozyme OxDD is not solely responsible for its reduced activity. When the loop of OxDC is altere d to similar effect, it has been shown that the catalytic efficiency and the capacity of the enzyme to maintain the reaction environment near the active site are not reduced in any meaningful capacity. The only significant effect that the mutation appears to exert on the enzyme is an inability to maintain its typical secondary structure and conformation, but even this does not appear to have a significant effect on the activity. Given that previous research had demonstrated that the removal of the loop inhi abilities do not have the same effect. Further study into the differences between OxDC and OxDD is necessary to truly uncover the cause behind their difference in activity. OUTLOOK While the primary focus of this thesis has been the decarboxylase activity of OxDC and OxDD, there also remains to be studied the oxidase activity of both iso zymes, which may
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30 provide additional insight into the effects of the alteration of the acti ve loop. OxDC exhibits oxidase activity in about 0.2% of turnovers, and other loop mutations have shown that a decrease in decarboxylase activity can lead to a corresponding increase in oxidase activity, though the reverse behavior remains to be tested fo r OxDD. 2 3 Testing of the oxidase activity of the N163F mutant will be able to confirm if, despite its negligible effect on decarboxylase activity, it has any effect on the oxidase activity of OxDC. Performing the same tests on the F171N and F171Y mutants w ould reveal if they lowered the oxidase activity of OxDD by increasing its decarboxylase activity, and give further insight into the relationship between the two cataly sis reactions of this enzyme. Additionally, the results of the Native PAGE and CD spectr a for the N163F mutant merit further and more precise investigation in the form of x ray crystallography. If the Native PAGE results are to be believed, the N163F mutant demonstrate s activity despite primarily existing as a monomer, something which has not been seen before in the study of OxDC. Even discounting this as a potential error in the PAGE, the clear differences in the secondary structure of the N163F mutant from the wild ty pe indicate a clear change in the conformation of the enzyme, yet no sig nificant difference in activity has resulted. This fact alone merits further investigation into the structure of N 163F, in order to determine if it truly possesses a unique conformation and what the implications of its activity are.
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31 ABBREVIATIONS Asn or N, asparagine; BeStSel, Beta Structure Selection; Bis tris, 2 [Bis(2 hydroxyethyl)amino] 2 (hydroxymethyl)propane 1,3 diol; BMPO, 3,4 dihydro 2 methyl 1,1 dimethylethyl ester 2H pyrrole 2 carboxylic acid 1 oxide; CD, circular dichrois m; DNA, deoxyribonucleic acid; E. Coli , Escherichia Coli ; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; F171N, mutant which exchanges phenylalanine 171 in the isozyme of oxalate decarboxylase with asparagine; F171Y, mutant wh ich exchanges phenylalanine 171 in the isozyme of oxalate decarboxylase with tyrosine; FDH, formate dehydrogenase; Glu or E, glutamate; 1 H, Hydrogen 1; His, histidine; ICP MS, inductively coupled plasma mass spectroscopy; D 1 thiogalactopyranoside; KLD, kinase, ligase and Dpnl enzymes; LB, Luria Broth; Mn, Manganese; 14 N, Nitrogen 14; N163F, mutant which exchanges asparagine 163 in oxalate decarboxylase with phenylalanine; NAD, nicotinamide adenine dinucleotide; NCBI, National Center for Biotechnology Information; NEB, New England Biolabs; Ni NTA, nickel nitrilotriacetic acid; o PDA, o phenylenediamine; OxDC, o xalate decarboxylase ; OxDD, isozyme of o xala te decarboxylase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Phe or F, phenylalanine; Ser or S, serine; TAE, Tris, acetate, and EDTA; Thr or T, threonine; Tris, tris(hydroxymethyl)aminomethane; WT, wild type . ACKNOWLEDGEMNTS I would like to thank Dr. Alexander Angerhofer for his support and guidance during the completion of this project, especially given the circumstances of a global pandemic which could have easily derailed it. I would also like to thank Dr. Anthony Pastore, Dr. Alvaro Montoya , and
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32 all other former and current members of the Angerhofer Research Group for their assistance and advice is bringing this project together. REFERENCES 1. Zhu, W.; Reinhardt, L. A.; Richards, N. G. J. Second Shell Hydrogen Bond Impacts Transition State Structure in Bacillus Subtilis Oxalate Decarboxylase. Biochemistry 2018 , 57 (24), 3425 3432. https://doi.org/10.1021/acs.biochem.8b00214 . 2. Just, V . J.; Burrell, M. R.; Bowater, L.; McRobbie, I.; Stevenson, C. E. M.; Lawson, D. M.; Bornemann, S. The Identity of the Active Site of Oxalate Decarboxylase and the Importance of the Stability of Active Site Lid Conformations. Biochemical Journal 2007 , 407 (3), 397 406. https://doi.org/10.1042/BJ20070708 . 3. The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. 4. Pastore, A. J.; Teo, R. D.; Montoya, A.; Burg, M. J.; Twahir, U. T.; Bruner, S. D.; Beratan, D. N.; Angerhofer, A. Oxalate Decarboxylase Uses Electron Hole Hopping for Catalysis. Journal of Biological Chemistry 2021 , 297 (1), 100857. https://doi.org/10.1016/j.jbc.2021.100857 . 5. Tanner, A.; Bowater, L.; Fairhurst, S. A.; Bornemann, S. Oxalate Decarboxylase Requires Manganese and Dioxygen for Activity : OVEREXPRESSION AND CHARACTERIZATION OF BACILLUS SUBTILIS YvrK AND YoaN. J. Biol. Chem. 2001 , 276 (47), 43627 43634. https://doi.org/10.1074/jbc.M107202200 . 6. Albert, A .; Tiwari, V.; Paul, E.; Ganesan, D.; Ayyavu, M.; Kujur, R.; Ponnusamy, S.; Shanmugam, K.; Saso, L.; Govindan Sadasivam, S. Expression of Heterologous Oxalate Decarboxylase in HEK293 Cells Confers Protection against Oxalate Induced Oxidative Stress as a Th erapeutic Approach for Calcium Oxalate Stone Disease. Journal of Enzyme
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33 Inhibition and Medicinal Chemistry 2017 , 32 (1), 426 433. https://doi.org/10.1080/14756366.2016.1256884 . 7. Saylor, B. T.; Reinhardt, L. A.; Lu, Z.; Shukla, M. S.; Nguyen, L.; Cleland, W. W.; Angerhofer, A.; Allen, K. N.; Richards, N. G. J. A Structural Element That Facilitates Proton Coupled Electron Transfer in Oxalate Decarboxylase. Biochemis try 2012 , 51 (13), 2911 2920. https://doi.org/10.1021/bi300001q . 8. Basic Local Alignment Search Tool, Version 1.21.0, National Center for Biotechnology Information, https://blast.ncbi.nlm.nih.gov/Blast.cgi . 9. Imaram, W.; Saylor, B. T.; Centonze, C. P.; Richards, N. G. J.; Angerhofer, A. EPR Spin Trapping of an Oxalate Derived F ree Radical in the Oxalate Decarboxylase Reaction. Free Radical Biology and Medicine 2011 , 50 (8), 1009 1015. https://doi.org/10.1016/j.freeradbiomed.2011.01.023 . 10. Wurm, D. J.; Veiter, L.; Ulonska, S.; Eggenreich, B.; Herwig, C.; Spadiut , O. The E. Coli PET Expression System Revisited Mechanistic Correlation between Glucose and Lactose Uptake. Appl Microbiol Biotechnol 2016 , 100 (20), 8721 8729. https://doi.org/10.1007/s00253 016 7620 7 . 11. Drey, C. N. C.; Fruton, J. S. Metal Chelates of a Bis Imidazole * . Biochemistry 1965 , 4 (7), 1258 1263. https://doi.org/10.1021/bi00883a007 . 12. Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein Dye Binding. An alytical Biochemistry 1976 , 72 (1 2), 248 254. https://doi.org/10.1006/abio.1976.9999 . 13. Artiukhov, A. V.; Pometun, A. A.; Zubanova, S. A.; Tishkov, V. I.; Bunik , V. I. Advantages of Formate Dehydrogenase Reaction for Efficient NAD+ Quantification in
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34 Biological Samples. Analytical Biochemistry 2020 , 603 , 113797. https://doi.org/10.1016/j.ab.2020.113797 . 14. Schutte, H.; Flossdorf, J.; Sahm, H.; Kula, M. R. Purification and Properties of Formaldehyde Dehydrogenase and Formate Dehydrogenase from Candida Boidinii. Eur J Biochem 1976 , 62 (1), 151 160. https://doi.org/10.1111/j.1432 1033.1976.tb10108.x . 15. , M. Spin Trapping of Oxygen Free Radicals in Chemical and Biological Systems: New Traps, Radicals and Possibilities. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2008 , 69 (5), 1354 1366. https://doi.org/10.1016/j.saa.2007.09.047 . 16. Beta Structure Selection, ELTE Eötvös Loránd University , https://bestsel.elte.hu/index.php . 17. Johnson, K. A.; Goody, R. S. The Original Michaelis Constant: Translation of the 1913 Michaelis Menten Paper. Biochemistry 2011 , 50 (39), 8264 8269. https://doi.org/10.1021/bi201284u . 18. Pastore, A. J. Investigation for Electron Transfer and Optimal Decarboxylase Activity of Oxalate Decarboxylase. Dissertation, University of Florida, 2020. https://ufdc.ufl.edu/UFE0056833/00001 . 19. MacNair, M. L. Site Directed Mutagenesis of the OxDD Active Site Loop. Undergraduate Thesis, University of Florida, 2023. 20. Twahir, U. T.; Stedwell, C. N.; Lee, C. T.; Richards, N. G. J.; Polfer, N. C.; Angerhofer, A. Observation of Superoxide Production during Catalysis of Bacillus Subtilis Oxalate Decarboxylase at PH 4. Free Radical Biology and Medicine 2015 , 80 , 59 66. https://doi.org/10.1016/j.freeradbiomed.2014.12.012 .
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35 21. Bielski, B. H. J.; Cabelli, D. E.; Arudi, R. L.; Ross, A. B. Reactivity of HO 2 /O 2 Radicals in Aqueous Solution. Journal of Physical and Chemical Reference Data 1985 , 14 (4), 1041 1100. https://doi.org/10.1063/1.555739 . 22. Rosen, G. M.; Britigen , B. E.; Halpern, H. J.; Pou, S. Free Radicals: Biology and Detection by Spin Trapping ; Oxford University Press: 198 Madison Avenue, New York, New York 10016, 1999. 23. Burrell, M. R.; Just, V. J.; Bowater, L.; Fairhurst, S. A.; Requena, L.; Lawson, D. M.; Bor nemann, S. Oxalate Decarboxylase and Oxalate Oxidase Activities Can Be Interchanged with a Specificity Switch of up to 282 000 by Mutating an Active Site Lid , . Biochemistry 2007 , 46 (43), 12327 12336. htt ps://doi.org/10.1021/bi700947s .
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Form online: https://guides.uflib.ufl.edu/ld.php?content_id=65544805 Permission to Quote/Reproduce Copyrighted Material I (We), Anthony John Pastore , owners(s) of the copyright of the work known a s INVESTIGATION FOR ELECTRON TRANSFER AND OPTIMAL DECARBOXYLASE ACTIVITY OF OXALATE DECARBOXYLASE hereby authorize Lucas A. Cathe y to use the following material as part of their thesis/dissertation /project to be submitted to the Institutional Repository at the Univer sity of Florida. P age Inclusive Line Numbers Passages to be Quoted/Reproduced Table 4 2 : Comparison of Decarboxylase Activity with C383A and W T. Page 116. T his information in cludes steady state en zyme kinetics data that is relevant for Lucas thesis. Attention : Theses and Dissertations Project Manager UF Libraries Digital Services University of Florida P.O. Box 11700 3
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mods:abstract lang en Oxalate decarboxylase (OxDC) is a naturally-occurring enzyme which catalyzes the decarboxylation of the mono-anion of oxalic acid in acidic conditions, a mechanism which has potential applications in both industrial and medical fields if it can be optimized and understood. The enzyme also possesses a naturally occurring isozyme, OxDD, which performs identical function with half the activity in natural conditions, and by determining the source of this difference a deeper understanding of the function of OxDC and its relationship with this isozyme can be obtained. The structures of the two enzymes possess a major discrepancy in a flexible peptide loop near the active site which has been demonstrated to effect enzymatic activity, so through site-directed mutagenesis the peptide loop of OxDC was altered to resemble that of OxDD; an asparagine in the loop was replaced with phenylalanine, a larger and less polar amino acid. Though it was expected that this alteration would result in a reduction in activity for the mutant as compared to the wild type (WT) of OxDC, testing has demonstrated that the inhibition of the peptide loop in this manner actually produces negligible changes in the overall function of the enzyme, though it does produce some structural differences. Given these results, it can be concluded that some other difference between the two isozymes is responsible for the greater activity of OxDC.
mods:accessCondition Copyright Lucas Aedán Cathey. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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