Sarcosine Oxidase Research Articles

Sarcosine Catabolism in Pseudomonas aeruginosa Is Transcriptionally Regulated by SouR

Sarcosine (N-methylglycine) is present in many environments inhabited by pseudomonads and is likely most often encountered as an intermediate in the metabolism of choline, carnitine, creatine, and glyphosate. While the enzymology of sarcosine metabolism has been relatively well studied in bacteria, the regulatory mechanisms governing catabolism have remained largely unknown. We previously determined that the sarcosine-catabolic (sox) operon of Pseudomonas aeruginosa is induced by the AraC family regulator GbdR in response to glycine betaine and dimethylglycine. However, induction of these genes was still observed in response to sarcosine in a gbdR deletion mutant, indicating that an independent sarcosine-responsive transcription factor also acted at this locus. Our goal in this study was to identify and characterize this regulator. Using a transposon-based genetic screen, we identified PA4184, or SouR (sarcosine oxidation and utilization regulator), as the sarcosine-responsive regulator of the sox operon, with tight induction specificity for sarcosine. The souR gene is required for appreciable growth on sarcosine as a carbon and nitrogen source. We also characterized the transcriptome response to sarcosine governed by SouR using microarray analyses and performed electrophoretic mobility shift assays to identify promoters directly regulated by the transcription factor. Finally, we characterized PA3630, or GfnR (glutathione-dependent formaldehyde neutralization regulator), as the regulator of the glutathione-dependent formaldehyde detoxification system in P. aeruginosa that is expressed in response to formaldehyde released during the catabolism of sarcosine. This study expands our understanding of sarcosine metabolic regulation in bacteria through the identification and characterization of the first known sarcosine-responsive transcriptional regulator. The Pseudomonas aeruginosa genome encodes many diverse metabolic pathways, yet the specific transcription regulators controlling their expression remain mostly unknown. Here, we used a genetic screen to identify the sarcosine-specific regulator of the sarcosine oxidase operon, which we have named SouR. SouR is the first bacterial regulator shown to respond to sarcosine, and it is required for growth on sarcosine. Sarcosine is found in its free form and is also an intermediate in the catabolic pathways of glycine betaine, carnitine, creatine, and glyphosate. The similarity of SouR to the regulators of carnitine and glycine betaine catabolism suggests evolutionary diversification within this regulatory family to allow response to structurally similar but physiologically distinct ligands.

Various instrumental approaches for determination of organic acids in wines

Biosensors based on lactate oxidase, sarcosine oxidase and mixture of fumarase and sarcosine oxidase were used for monitoring of organic acids in wine samples. Additionally, tartaric acid was determined by modified colorimetric method based on formation of the vanadate-tartrate complex. The above mentioned methods were used for the analysis of 31 wine samples and obtained data were compared with the results from capillary electrophoresis as a basic standard method. This comparison showed a certain degree of correlation between biosensors and capillary electrophoresis. The provided information pointed to the potential uses of biosensors in the field of winemaking.

On the Quest of Dioxygen by Monomeric Sarcosine Oxidase. A Molecular Dynamics Investigation.

It is reported here on random acceleration molecular dynamics (RAMD) simulations with the 2GF3 bacterial monomeric sarcosine oxidase (MSOX), O2 , and furoic acid in place of sarcosine, solvated by TIP3 H2 O in a periodic box. An external tiny force, acting randomly on O2 , accelerated its relocation, from the center of activation between residue K265 and the si face of the flavin ring of the flavin adenine dinucleotide cofactor, to the surrounding solvent. Only three of the four O2 gates previously described for this system along a composite method technique were identified, while two more major O2 gates were found. The RAMD simulations also revealed that the same gate can be reached by O2 along different pathways, often involving traps for O2 . Both the residence time of O2 in the traps, and the total trajectory time for O2 getting to the solvent, could be evaluated. The new quick pathways discovered here suggest that O2 exploits all nearby interstices created by the thermal fluctuations of the protein, not having necessarily to look for the permanent large channel used for uptake of the FADH cofactor. To this regard, MSOX resembles closely KijD3 N-oxygenase. These observations solicit experimental substantiation, in a long term aim at discovering whether gates and pathways for the small gaseous ligands inside the proteins are under Darwinian functional evolution or merely stochastic control operates.

Glutathione metabolism links FOXRED1 to NADH:ubiquinone oxidoreductase (complex I) deficiency: A hypothesis

FOXRED1 mutations result in complex I (NADH:ubiquinone oxidoreductase) deficiencies and Leigh syndrome (subacute necrotizing encephalomyelopathy). FOXRED1 is a mitochondrial flavoprotein related to N-methyl amino acid dehydrogenases. How is FOXRED1 required for the biogenesis of complex I? I present a hypothesis that suggests FOXRED1 catalytic activity as a sarcosine oxidase protects the developing fetus from oxidative stress during pregnancy. Loss of FOXRED1, coupled with protein, choline and/or folate-deficient diets results in the depletion of glutathione, the dysregulation of nitric oxide metabolism and the peroxynitrite-mediated inactivation of complex I.

A facile low-cost enzymatic paper-based assay for the determination of urine creatinine

Creatinine is one of many markers used to investigate kidney function. This paper describes a low-cost enzymatic paper-based analytical device (enz-PAD) for determining urine creatinine. The disposable dead volumes of creatinine enzyme reagents from an automatic analyser cassette were utilised. Whatman No. 3 paper was cut into long rectangular shapes (4×40mm2) on which the enzyme reagents, R1 and R2, were adsorbed in two consecutive regions. The assay was performed by immersing test strips into urine samples contained in microwells to allow creatinine in the sample to react with immobilised active ingredients and, then, traverse via capillary action to the detection area where chromogen products accumulated. The method is based on hydrogen peroxide (H2O2) formation via creatinine conversion using creatininase, creatinase, and sarcosine oxidase. The liberated H2O2 reacts with 4-aminophenazone and 2,4,6-triiodo-3-hydroxybenzoic acid to form quinoneimine with a pink-red colour at the detection zone. The linear range of the creatinine assay was 2.5–25mgdL−1 (r2=0.983), and the detection limit was 2.0mgdL−1. The colorimetric enz-PAD for the creatinine assay was highly correlated with a conventional alkaline picrate method when real urine samples were evaluated (r2=0.977; n=40). This simple and nearly zero-cost paper-based device provides a novel alternative method for screening urinary creatinine and will be highly beneficial for developing countries.

Novel integration strategy coupling codon and fermentation optimization for efficiently enhancing sarcosine oxidase (SOX) production in recombinant Escherichia coli.

Sarcosine oxidase (SOX) was an important diagnostic enzyme in the renal function examination. An integrated strategy coupling codon and fermentation optimization was firstly proposed for improving SOX production from recombinant E. coli in 3-L fermentor. The expression suppression (gene phase) and poor balance between SOX expression and cell growth (fermentation phase) in the traditional SOX production were respectively improved by the multiple strategies. Based on the codon bias, the expression suppression was weakened via codon optimization and SOX activity reached 1,521 U/L. The induction toxicity was reduced with the optimal induction condition and SOX production increased to 4,015 U/L. Based on the kinetic analysis of μ x and μ p , a better balance between cell growth and expression was achieved by the two-stage pH-stat control strategy. The SOX activity was further improved to 8,490 U/L and fermentation cycle was also significantly shortened from 44 to 32 h. The substrate inhibition was weakened with a constant feeding fed-batch. With the assistance of integrated strategy, the activity and productivity reached 12,466 U/L and 389.6 U/(L h), respectively, or 3.1-fold and 4.3-fold of the uncontrolled fermentation. The strategy would be also useful in the industrial application of other similar enzymes.

Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic structures.

Zero-mode waveguides (ZMW) have the potential to be powerful confinement tools for studying electron transfer dynamics at single molecule occupancy conditions. Flavin mononucleotide contains an isoalloxazine chromophore, which is fluorescent in the oxidized state (FMN) while the reduced state (FMNH2) exhibits dramatically lower light emission, i.e. a dark-state. This allows fluorescence emission to report the redox state of single FMN molecules, an observation that has been used previously to study single electron transfer events in surface-immobilized flavins and flavoenzymes, e.g. sarcosine oxidase, by direct wide-field imaging of ZMW arrays. Single molecule electron transfer dynamics have now been extended to the study of freely diffusing molecules using fluorescence measurements of Au ZMWs under single occupancy conditions. The Au in the ZMW serves both as an optical cladding layer and as the working electrode for potential control, thereby accessing single molecule electron transfer dynamics at μM concentrations. Consistent with expectations, the probability of observing single reduced molecules increases as the potential is scanned negative, E(appl) < E(eq), and the probability of observing emitting oxidized molecules increases at E(appl) > E(eq). Different single molecules exhibit different electron transfer properties as reflected in the position of E(eq) and the distribution of E(eq) among a population of FMN molecules. Two types of actively-controlled electroluminescence experiments were used: chronofluorometry experiments, in which the potential is alternately stepped between oxidizing and reducing potentials, and cyclic potential sweep fluorescence experiments, analogous to cyclic voltammetry, these latter experiments exhibiting a dramatic scan rate dependence with the slowest scan rates showing distinct intermediate states that are stable over a range of potentials. These states are assigned to flavosemiquinone species that are stabilized in the special environment of the ZMW nanopore.

Markovian Milestoning for Computing Entry, Exit, and Internal Diffusion Rates of Ligands in Proteins

Measuring diffusion rates of ligands plays a key role in understanding the kinetic processes inside proteins. For example, although many molecular simulation studies have reported free energy barriers to infer rates for CO diffusion in myoglobin (Mb), they typically do not include direct calculation of diffusion rates because of the long simulation times needed to infer these rates with statistical accuracy. We show in this talk how to apply Markovian milestoning along minimum free-energy pathways to calculate diffusion rates of both CO inside Mb and O2 inside monomeric sarcosine oxidase (MSOX). In Markovian milestoning, one partitions a suitable reaction coordinate space into regions and performs restrained molecular dynamics in each region to accumulate kinetic statistics that, when assembled across regions, provides an estimate of the mean first-passage time between states. We show here that the milestones can be chosen from a Voronoi tessellation defined by discrete centers taken from minimum free-energy pathways computed using the single-sweep reconstruction method. In the case of CO/Mb, we find that, although simulations predict the existence of many potential portals that connect the solvent phase with the distal pocket (DP, the site of CO attachment to the heme iron), the so-called “histidine gate” pathway is kinetically dominant for both CO entry and exit by approximately a factor of ten, in qualitative agreement with existing experimental data. Our calculations also semi-quantitatively agree with experiment, predicting a 60-ns mean exit time for CO from the DP and a mean entry time of 50 microsec under CO-saturating conditions. The Markovian milestoning approach seems from these results to be a promising way to estimate biomolecule-related transition rates, and ongoing work involves using it to time conformational changes.

Analysis of water channels by molecular dynamics simulation of heterotetrameric sarcosine oxidase.

A precise 100-ns molecular dynamics simulation in aquo was performed for the heterotetrameric sarcosine oxidase bound with a substrate analogue, dimethylglycine. The spatial region including the protein was divided into small rectangular cells. The average number of the water molecules locating within each cell was calculated based on the simulation trajectory. The clusters of the cells filled with water molecules were used to determine the water channels. The narrowness of the channels, the average hydropathy indices of the residues of the channels, and the number of migration events of water molecules through the channels were consistent with the selective transport hypothesis whereby tunnel T3 is the pathway for the exit of the iminium intermediate of the enzyme reaction.

Sarcosine oxidase composite screen-printed electrode for sarcosine determination in biological samples

As the prostate cancer (PCa) progresses, sarcosine levels increase both in tumor cells and urine samples, suggesting that this metabolite measurements can help in the creation of non-invasive diagnostic methods for this disease. In this work, a biosensor device was developed for the quantification of sarcosine via electrochemical detection of H2O2 (at 0.6V) generated from the catalyzed oxidation of sarcosine. The detection was carried out after the modification of carbon screen printed electrodes (SPEs) by immobilization of sarcosine oxidase (SOX) on the electrode surface. The strategies used herein included the activation of the carbon films by an electrochemical step and the formation of an NHS/EDAC layer to bond the enzyme to the electrode, the use of metallic or semiconductor nanoparticles layer previously or during the enzyme immobilization. In order to improve the sensor stability and selectivity a polymeric layer with extra enzyme content was further added. The proposed methodology for the detection of sarcosine allowed obtaining a limit of detection (LOD) of 16nM, using a linear concentration range between 10 and 100nM. The biosensor was successfully applied to the analysis of sarcosine in urine samples.

Oxygen Pathways and Allostery in Monomeric Sarcosine Oxidase via Single-Sweep Free-Energy Reconstruction.

Monomeric sarcosine oxidase (MSOX) is a flavoprotein D-amino acid oxidase with reported sarcosine and oxygen activation sites on the re and si faces of the flavin ring, respectively. O2 transport routes to the catalytic interior are not well understood and are difficult to ascertain solely from MSOX crystal structures. A composite free-energy method known as single-sweep is used to map and thermodynamically characterize oxygen sites and routes leading to the catalytically active Lys265 from the protein surface. The result is a network of pathways and free energies within MSOX illustrating that oxygen can access two free-energy minima on the re face of the reduced flavin from four separate solvent portals. No such minimum is observed on the si face. The pathways are geometrically similar for three major states of the enzyme: (1) apo with a closed flavin cleft, (2) apo with an open flavin cleft, and (3) inhibitor-bound with a closed flavin cleft. Interestingly, free energies along these transport pathways display significantly deeper minima when the substrate-mimicking inhibitor 2-furoic acid is bound at the sarcosine site, even at locations far from this site. This suggests a substrate-dependent allosteric modulation of the kinetics of O2 transport from the solvent to the active site.

Development of a Stable, Multi-Use Creatinine Biosensor with Extended Use Life

Development of a Stable, Multi-Use Creatinine Biosensor with Extended Use Life

Photocurrent Measurements at Quantum Dot Electrodes for Bioanalytical Applications

Quantum dots (QDs) allow the generation of electron-hole pairs upon illumination. When these particles are fixed to an electrode a photocurrent can be generated. This allows their use as a light-switchable layer on the surface. The QDs can not only exchange electrons with the electrode, but can also show reactions with donor or acceptor compounds in solution. This provides access to the construction of signal chains starting from an analyte molecule to be detected.The direction and magnitude of the photocurrent depend on several factors such as electrode polarization, solution pH and composition. These defined dependencies have been evaluated with respect to the combination of QD-electrodes with enzyme reactions for sensorial purpose [1,2].Thus, it has been found that the cofactor of many dehydrogenase-based reactions NADH can be oxidized at the illuminated QDs without applying high potentials. This provides the basis for a coupling strategy with the enzyme glucose dehydrogenase for the analysis of glucose concentrations in solution by photocurrent measurements.CdSe/ZnS-QD-modified electrodes can also be used to follow enzymatic reactions in solution based on their capability to reduce molecular oxygen while being excited [3]. In order to develop a photoelectrochemical biosensor, glucose oxidase (GOD) is immobilized on the QD-electrode. One immobilization strategy applies the layer-by-layer-technique of GOD and a polyelectrolyte (polyallylamine hydrochloride). Photocurrent measurements of such a sensor show a clear concentration dependent behavior for millimolar glucose concentrations. Sensitivity can be influenced by the enzyme concentration and the number of layers deposited. The principle of combining QD electrodes with a layered protein architecture and light-triggered read-out can also be transferred to other enzymes such sarcosine oxidase and sarcosine detection. The sensing properties of quantum dot electrodes can be significantly influenced by additional nanoparticles giving access to the detection of other substances, but also by immobilizing multiple layers of the QDs.In another direction of research it can be demonstrated that direct electron transfer from excited quantum dots can be achieved with the redox protein cytochrome c provided the QD surface is properly modified. This allows the detection of the protein in different concentrations and redox states, but also of interaction partners which show a defined reaction with the protein. Examples are the cyt c reduction by superoxide radicals resulting in an anodic photocurrent and the cyt c oxidation by nitrate reductase in the presence of nitrate resulting in a cathodic photocurrent .[1] Ch. Stoll, S. Kudera, W.J. Parak, F. Lisdat, SMALL 2 (6) (2006) 741-743).[2] K. Schubert, W. Khalid, Z. Yue, W. Parak, F. Lisdat, Langmuir 26 (2) (2010) 1395-1400[3] J. Tanne, D. Schäfer, W. Khalid, W. J. Parak, F. Lisdat, Analytical Chemistry 83 (20) (2011) 7778–7785

Mechanistic and computational studies on C‐N bond oxidation in D‐amino acids catalyzed by D‐arginine dehydrogenase Y53F and Y249F (584.4)

Pseudomonas aeruginosa D‐arginine dehydrogenase (PaDADH) is a flavin dependent enzyme that can be classified as a member of D‐amino acid oxidase, sarcosine oxidase, and related enzyme families. It catalyzes the oxidation of D‐amino acids except D‐aspartate, D‐glutamate and glycine, to imino acids. Mechanistic details of C‐N bond oxidations of D‐amino acids by PaDADH are still elusive. Multiple mechanistic possibilities such as carbanion, hydride transfer and polar nucleophilic mechanisms could be considered. Previous work on PaDADH showed that NH and CH bond cleavages during amine oxidation are asynchronous with no further details available on the chemical mechanism. Here, we mutated two conserved active site residues Y53 and Y249 to phenylalanine in an attempt to reveal the mechanistic details of amine oxidation via pH profiles and deuterium isotope effects on rapid reaction kinetics. To further minimize mechanistic possibilities, QM/MM methodology was used to probe the relative local electrophilicity of key FAD sites within PaDADH active site. Our results are consistent with D‐amino acid oxidation occurring through the direct transfer of a hydride ion from the substrate to FAD without stabilization of reaction intermediates or FAD‐derived adducts, in which the active site tyrosines do not have a direct role in catalysis in PaDADH.Grant Funding Source: NSF MCB‐1121695 (G.G.) and a GSU Molecular Basis for Disease Fellowship (S.G.).

Efficient improvement on stability of sarcosine oxidase via poly-lysine modification on enzyme surface

A novel modification method was proposed for improving the stability of sarcosine oxidase. In this process, sarcosine oxidase surface was efficiently linked with poly-lysine (poly-Lys) covalently after activation with N-ethyl-N′-3-dimethylaminopropyl carbodiimide (EDC); the optimal conditions for this reaction were also investigated. The molar ratios of enzyme-COOH to EDC and enzyme-COOH to poly-Lys-NH2 were 1:2 and 1:50, respectively, while the optimal reaction pH was 7.0. The covalently binding of poly-Lys onto enzyme surface was confirmed by mass spectrum (MS) and Fourier transform infrared spectroscopy (FTIR). The catalytic kinetic parameters (Km and Vmax) of modified enzyme were determined as 47.94mM and 0.157μmol/min, respectively. Moreover, compared to the native enzyme, the pH, thermal and storage stabilities of modified sarcosine oxidase were significantly improved. More than 90% of initial activity of modified enzyme was maintained at a broad pH range from 5.0 to 10.0. Most activity of modified enzyme could be detected after being incubated at 60°C for 10min. The storage stability was enhanced ∼12-fold after being stored at 37°C for 7 days. The novel modification was highly efficient for improving the stability of sarcosine oxidase and might be a good reference for other similar enzymes.

Sarcosine Oxidase Encapsulated Polyvinyl Alcohol-Silica-AuNP Hybrid Films for Sarcosine Sensing Electrochemical Bioelectrode

The amperometric response of sarcosine was measured in aqueous media containing ferrocene monocarboxylic acid using the redox enzyme sarcosine oxidase (SOD) immobilized in a composite material based on polyvinyl alcohol (PVA) and partially prehydrolyzed tetraethyl orthosilicate (pphTEOS) at the surface of “in-house” fabricated graphite electrodes. For comparison, separate electrodes consisting of silver and gold nanoparticles (Ag/AuNPs) embedded in the PVA-pphTEOS matrix was prepared employing a novel sol-gel process based on the in situ chemical reduction of Ag or Au ions using PVA both, as a reducing agent and stabilizer. The analytical performance of the enzyme electrodes was studied in terms of linear ranges, sensitivities, response times, limits of detection, reproducibility and stability.

1P068 4量体型サルコシン酸化酵素の分子動力学シミュレーション : 反応物と生成物の輸送経路の解明(01D. 蛋白質 : 機能,ポスター,第52回日本生物物理学会年会(2014年度))

1P068 4量体型サルコシン酸化酵素の分子動力学シミュレーション : 反応物と生成物の輸送経路の解明(01D. 蛋白質 : 機能,ポスター,第52回日本生物物理学会年会(2014年度))

Novel affinity purification of monomeric sarcosine oxidase expressed in Escherichia coli

An efficient affinity-purification protocol for Bacillus monomeric sarcosine oxidase (SOX) expressed in Escherichia coli BL21 (DE3) was developed. 4-Aminopyrrole-2-carboxylic acid was chosen as the affinity ligand, which was coupled with Sepharose CL 4B via spacers composed of epichlorohydrin and ethylenediamine. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture monomeric SOX. The purified SOX was 94 and 96% pure when analyzed on an HPLC Vydac C8 column and reducing SDS-PAGE. Meanwhile, the recoveries of typical SOX activity and protein were 90.8 and 37.5%, respectively, which were higher than other reported traditional protocols. Reducing SDS-PAGE analysis revealed that the enzyme was a single polypeptide with the mass of ~46 kDa. The desorption constant Kd and theoretical maximum absorption Qmax were 35 μg/mL and 52.7 mg/g, respectively, in absorption analysis. All results indicated that the method would be of great potential for purifying monomeric SOX on an industrial scale.

An inter-order horizontal gene transfer event enables the catabolism of compatible solutes by Colwellia psychrerythraea 34H

Colwellia is a genus of mostly psychrophilic halophilic Gammaproteobacteria frequently isolated from polar marine sediments and sea ice. In exploring the capacity of Colwellia psychrerythraea 34H to survive and grow in the liquid brines of sea ice, we detected a duplicated 37 kbp genomic island in its genome based on the abnormally high G + C content. This island contains an operon encoding for heterotetrameric sarcosine oxidase and is located adjacent to several genes used in the serial demethylation of glycine betaine, a compatible solute commonly used for osmoregulation, to dimethylglycine, sarcosine, and glycine. Molecular clock inferences of important events in the adaptation of C. psychrerythraea 34H to compatible solute utilization reflect the geological evolution of the polar regions. Validating genomic predictions, C. psychrerythraea 34H was shown to grow on defined media containing either choline or glycine betaine, and on a medium with sarcosine as the sole organic source of carbon and nitrogen. Growth by 8 of 9 tested Colwellia species on a newly developed sarcosine-based defined medium suggested that the ability to catabolize glycine betaine (the catabolic precursor of sarcosine) is likely widespread in the genus Colwellia. This capacity likely provides a selective advantage to Colwellia species in cold, salty environments like sea ice, and may have contributed to the ability of Colwellia to invade these extreme niches.

Sarcosine metabolism in Haemonchus contortus and Teladorsagia circumcincta

Sarcosine (N-methylglycine) is an intermediate in glycine degradation and can also be synthesised from glycine in mammals. Sarcosine metabolism in Haemonchus contortus and Teladorsagia circumcincta differed from that of mammals in that creatinase activity was present and sarcosine was demethylated only by sarcosine oxidase (SOX) and not by sarcosine dehydrogenase (SDH). The mean SOX activity was 30 nmolmin(-1)mg(-1) protein in homogenates of L3 and adult worms of both parasites and the apparent Km for sarcosine was 1.1 mM. Addition of 2 mM Cd(2+) inhibited activity by 30%. There was no SDH activity with either NAD(+) or NADP(+) as co-factor. Mean creatinase activity in L3 T. circumcincta and adult worms of both species was 31±6 nmolmin(-1)mg(-1) protein, but was undetectable in L3 H. contortus. Activity was inhibited by up to 70% by Cu(2+), Fe(2+), Fe(3+) and Zn(2+). Possessing creatinase would allow host creatine to be incorporated into amino acids by the parasites.