Apparent Kcat Values Research Articles

A Simple and Sensitive Approach for Real‐Time Sensing of Enzymatically Catalyzed Hydrogelation

AbstractDespite the critical role of hydrogels in material science and biotechnology, current methods for analyzing their formation lack real‐time monitoring and require complex sample preparation and instrumentation. In this work, an innovative methodology is introduced for the real‐time analysis of enzymatically catalyzed hydrogelation. Electrochemical impedance spectroscopy (EIS) coupled with interdigitated electrodes (IDEs) to sense and transduce the gelation reaction of model precursor carboxymethyl cellulose‐tyramine (CMC‐TA) conjugates that undergoes enzymatic cross‐linking by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Real‐time monitoring involves single‐frequency analyses at 3 × 105 Hz, where the measured impedance consists solely of a resistive component, and the admittance equates to solution conductance. The gelation trajectories for all tested enzymatically cross‐linked hydrogel component combinations are determined by substituting the conductance data in the modified Michaelis–Menten kinetic model. Specifically, for CMC‐TA cross‐linked by HRP, the authors calculate apparent KM and kcat values of 82.1 µM and 95.5 s−1, respectively. These findings are further validated through rheological characterization, including oscillatory shear measurements and microrheology. Overall, this research paves the way for a streamlined, accurate, and cost‐effective approach to controllable enzymatically initiated hydrogel synthesis, enhancing their successful application in various fields ranging from material science to biotechnology.

Purification, biochemical characterization, and biotechnological applications of a multifunctional enzyme from the Thermoascus aurantiacus PI3S3 strain

The filamentous Thermoascus aurantiacus fungus characterized by its thermophilic nature, is recognized as an exceptional producer of various enzymes with biotechnological applications. This study aimed to explore biotechnological applications using polygalacturonase (PG) derived from the Thermoascus aurantiacus PI3S3 strain. PG production was achieved through submerged fermentation and subsequent purification via ion-exchange chromatography and gel filtration methods. The crude extract exhibited a diverse spectrum of enzymatic activities including amylase, cellulase, invertase, pectinase, and xylanase. Notably, it demonstrated the ability to hydrolyze sugarcane bagasse biomass, corn residue, and animal feed. The purified PG had a molecular mass of 36 kDa, with optimal activity observed at pH 4.5 and 70 °C. The activation energy (Ea) was calculated as 0.513 kJ mol−1, highlighting activation in the presence of Ca2+. Additionally, it displayed apparent Km, Vmax, and Kcat values of at 0.19 mg mL−1, 273.10 U mL−1, and 168.52 s−1, respectively, for hydrolyzing polygalacturonic acid. This multifunctional PG exhibited activities such as denim biopolishing, apple juice clarification, and demonstrated both endo- and exo-polygalacturonase activities. Furthermore, it displayed versatility by hydrolyzing polygalacturonic acid, carboxymethylcellulose, and xylan. The T. aurantiacus PI3S3 multifunctional polygalacturonase showed heightened activity under acidic pH, elevated temperatures, and in the presence of calcium. Its multifunctional nature distinguished it from other PGs, significantly expanding its potential for diverse biotechnological applications.

CRABPs Alter all-trans-Retinoic Acid Metabolism by CYP26A1 via Protein-Protein Interactions.

Cellular retinoic acid binding proteins (CRABP1 and CRABP2) bind all-trans-retinoic acid (atRA), the active metabolite of vitamin A, with high affinity. CRABP1 and CRABP2 have been shown to interact with the atRA-clearing cytochrome P450 enzymes CYP26B1 and CYP26C1 and with nuclear retinoic acid receptors (RARs). We hypothesized that CRABP1 and CRABP2 also alter atRA metabolism and clearance by CYP26A1, the third key atRA-metabolizing enzyme in the CYP26 family. Based on stopped-flow experiments, atRA bound CRABP1 and CRABP2 with Kd values of 4.7 nM and 7.6 nM, respectively. The unbound atRA Km values for 4-OH-atRA formation by CYP26A1 were 4.7 ± 0.8 nM with atRA, 6.8 ± 1.7 nM with holo-CRABP1 and 6.1 ± 2.7 nM with holo-CRABP2 as a substrate. In comparison, the apparent kcat value was about 30% lower (0.71 ± 0.07 min−1 for holo-CRABP1 and 0.75 ± 0.09 min−1 for holo-CRABP2) in the presence of CRABPs than with free atRA (1.07 ± 0.08 min−1). In addition, increasing concentrations in apo-CRABPs decreased the 4-OH-atRA formation rates by CYP26A1. Kinetic analyses suggest that apo-CRABP1 and apo-CRABP2 inhibit CYP26A1 (Ki = 0.39 nM and 0.53 nM, respectively) and holo-CRABPs channel atRA for metabolism by CYP26A1. These data suggest that CRABPs play a critical role in modulating atRA metabolism and cellular atRA concentrations.

Looking into a highly thermostable and efficient recombinant manganese-catalase from Geobacillusthermopakistaniensis

Catalases, heme or non-heme, are catalysts that decompose hydrogen peroxide. Among them, non-heme or manganese-catalases have been studied from limited organisms. We report here heterologous production of a manganese-catalase, Cat-IIGt, previously annotated as a hypothetical protein, from a thermophilic bacterium Geobacillus thermopakistaniensis. Recombinant Cat-IIGt, produced as inactive inclusion bodies in Escherichia coli, was solubilized and refolded into a soluble and highly active form. Sequence homology, absorption spectra, resistance to sodium azide inhibition and activation by Mn2+ indicated that it was a manganese-catalase. Metal analysis revealed the presence of ∼2 Mn2+ and ∼2 Ca2+ per subunit of Cat-IIGt. Recombinant Cat-IIGt exhibited highest activity at pH 10.0 and 70°C. The enzyme was highly active with a specific activity of 40,529μmolmin-1 mg-1. The apparent Km and kcat values were 75mM and 1.5× 104 s-1 subunit-1, respectively. Recombinant Cat-IIGt was highly thermostable with a half-life of 30min at 100°C. The structural attributes of Cat-IIGt, including the metal and substrate binding residues, were predicted by homology modeling and molecular docking studies. High activity and thermostability and alkaline nature make Cat-IIGt a potential candidate for textile and paper processing industries.

Heterologous expression and exploration of the enzymatic properties of the carbaryl hydrolase CarH from a newly isolated carbaryl-degrading strain

Carbaryl is the representative of carbamate insecticide. As an acetylcholinesterase inhibitor, it poses potential threat to humans and other non-target organisms. Agrobacterium sp. XWY-2, which could grow with carbaryl as the sole carbon source, was isolated and characterized. The carH gene, encoding a carbaryl hydrolase, was cloned from strain XWY-2 and expressed in Escherichia coli BL21 (DE3). CarH was able to hydrolyze carbamate pesticides including carbaryl, carbofuran, isoprocarb, propoxur and fenobucarb efficiently, while it hydrolyzed oxamyl and aldicarb poorly. The optimal pH of CarH was 8.0 and the optimal temperature was 30 ℃. The apparent Km and kcat values of CarH for carbaryl were 38.01 ± 2.81 μM and 0.33 ± 0.01 s−1, respectively. The point mutation experiment demonstrated that His341, His343, His346, His416 and D437 are the key sites for CarH to hydrolyze carbaryl.

Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus.

N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 μM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 μM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.

Structural and functional analyses of a novel manganese-catalase from Bacillus subtilis R5

Catalases catalyze the decomposition of hydrogen peroxide into water and oxygen. Limited reports are available on characterization of manganese-catalases. We describe here molecular cloning and expression in Escherichia coli of a putative manganese-catalase gene from mesophilic bacterium, Bacillus subtilis R5. The gene product, CatBsu, produced as a soluble protein, was purified to apparent homogeneity and biochemically characterized. The absorption spectra and nonsignificant inhibition by sodium azide indicated that it is a manganese-catalase. The protein was in homohexameric form in solution, with a subunit molecular weight of 30 kDa, containing ~2 Mn2+ and ~1 Ca2+ per subunit. CatBsu showed highest activity at pH 8.0 and 55 °C. It was found to be highly active with a specific activity of 25,290 μmol min−1 mg−1 and apparent Km and kcat values of 98 mM and 1.27 × 104 s−1 subunit−1, respectively. Although from a mesophilic source, it exhibited a half-life of 2 h at 80 °C. Furthermore, the active site and metal binding residues in CatBsu were predicted by homology modelling and molecular docking. To the best of our knowledge, this is the first characterization of a manganese-catalase from genus Bacillus.

Identification of the key amino acid sites of the carbofuran hydrolase CehA from a newly isolated carbofuran-degrading strain Sphingbium sp. CFD-1.

A novel carbofuran-degrading strain CFD-1 was isolated and preliminarily identified as Sphingbium sp. This strain was able to utilize carbofuran as the sole carbon source for growth. The carbofuran hydrolase gene cehA was cloned from strain CFD-1 and expressed in Escherichia coli. CehA could hydrolyze carbamate pesticides including carbofuran and carbaryl efficiently, while it showed poor hydrolysis ability against isoprocarb, propoxur, oxamyl and aldicarb. CehA displayed maximal enzymatic activity at 40 °C and pH 7.0. The apparent Km and Kcat values of CehA for carbofuran were 133.22 ± 5.70 μM and 9.48 ± 0.89 s-1, respectively. The site-directed mutation experiment showed that His313, His315, His453 and His495 played important roles in the hydrolysis of carbofuran by CehA. Furthermore, the sequence of cehA is highly conserved among different carbofuran-degrading strains, and there are mobile elements around cehA, indicating that it may be transferred horizontally between different strains.

Biochemical characterization and structural analysis of ulvan lyase from marine Alteromonas sp. reveals the basis for its salt tolerance

Marine macroalgae have gained considerable attention as renewable biomass sources. Ulvan is a water-soluble anionic polysaccharide, and its depolymerization into fermentable monosaccharides has great potential for the production of bioethanol or high-value food additives. Ulvan lyase from Alteromonas sp. (AsPL) utilizes a β-elimination mechanism to cleave the glycosidic bond between rhamnose 3-sulfate and glucuronic acid, forming an unsaturated uronic acid at the non-reducing end. AsPL was active in the temperature range of 30–50 °C and pH values ranging from 7.5 to 9.5. Furthermore, AsPL was found to be halophilic, showing high activity and stability in the presence of up to 2.5 M NaCl. The apparent Km and kcat values of AsPL are 3.19 ± 0.37 mg mL−1 and 4.19 ± 0.21 s−1, respectively. Crystal structure analysis revealed that AsPL adopts a β-propeller fold with four anti-parallel β-strands in each of the seven propeller blades. The acid residues at the protein surface and two Ca2+ coordination sites contribute to its salt tolerance. The research on ulvan lyase has potential commercial value in the utilization of algal resources for biofuel production to relieve the environmental burden of petrochemicals.

Biotechnological potential of an exo-polygalacturonase of the new strain Penicillium janthinellum VI2R3M: biochemical characterization and clarification of fruit juices.

The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M. The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion-exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0-5·0. The apparent Km , Vmax and Kcat values for hydrolyzing polygalacturonic acid were 2·56mgml-1 , 163·1Umg-1 and 277s-1 respectively. The polygalacturonase presented exo activity and was activated by Mg2+ . The juices treated with polygalacturonase presented increases in transmittance with reduction in colour. The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo-polygalacturonase induced by agro-industrial residues, with excellent activity and stability in acidic pH and at 50°C. The use of agro-industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application.

A novel thermostable d-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii strain YA

d-Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining d-amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specificity. In this study, we isolated the thermophilic fungus Rasamsonia emersonii strain YA, which can grow on several d-amino acids as the sole nitrogen source, from a compost and characterized DAAO (ReDAAO) of the fungus. ReDAAO expressed in Escherichia coli exhibited significant oxidase activity against various neutral and basic d-amino acids, in particular hydrophobic d-amino acids. In addition, the enzyme also significantly acted on cephalosporin C, a starting material for semisynthetic antibiotics, and d-Glu, a general substrate for d-aspartate oxidase but not for DAAO, showing its unique and practically useful substrate specificity. The apparent kcat and Km values of the enzyme toward good substrates were comparable to those of higher catalytic fungal DAAOs, and the thermal stability (T50 value of ~60 °C) was comparable to that of a thermophilic bacterial DAAO and significantly higher than that of other eukaryotic DAAOs. These results highlight the great potential of ReDAAO for use in practical applications.

A highly stable manganese catalase from Geobacillus thermopakistaniensis: molecular cloning and characterization.

Catalases, heme or manganese, are efficient biocatalysts that split hydrogen peroxide into water and oxygen. We have cloned a manganese catalase from thermophilic bacterium, Geobacillus thermopakistaniensis, and expressed the corresponding gene in Escherichia coli. The gene product, CatGt, was synthesized in E. coli as inactive inclusion bodies. Solubilization and refolding of the inclusion bodies resulted in highly active CatGt with a specific activity of 18,521μmolmin-1mg-1. The refolded protein exhibited apparent Km and kcat values of 260mM and 10,360s-1 subunit-1, respectively. It exhibited a half-life of 1h at 100°C. The unique features of CatGt are its high activity and thermostability. These features make it a valuable catalyst for industrial applications. To the best of our knowledge, CatGt is the most thermostable catalases characterized to date.

Biochemical characterization of a highly active ADP-dependent phosphofructokinase from Thermococcus kodakarensis

The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK0376, annotated as ADP-dependent phosphofructokinase belonging to pfkC family. The encoding gene was expressed in Escherichia coli and the gene product was characterized. The recombinant protein was produced in soluble and active form. Phosphofructokinase activity of TK0376 was metal-ion dependent and the highest activity (5090μmolmin-1 mg-1) was found in the presence of Co2+ followed by Mg2+ (3280μmolmin-1 mg-1) at 90°C and pH 7.5. TK0376 preferred ADP as phosphoryl donor, however, it could be replaced by ATP but with a 5-fold lower activity. It catalyzed the phosphorylation of fructose 6-phosphate and dephosphorylation of fructose 1,6-bisphosphate. In addition, it was able to phosphorylate glucose and nucleosides but with a much lower rate compared to that of fructose 6-phosphate. The apparent kcat and Km values against fructose 6-phosphate were 4238 s-1 and 0.74mM, respectively. The rate of dephosphorylation of fructose 1,6-bisphosphate was 3-times lower at 50°C than the phosphorylation of fructose 6-phosphate. Similarly, the rate of phosphorylation of glucose was 450-fold lower than that of fructose 6-phosphate. Phosphofructokinase activity was not allosterically regulated, but it was slightly enhanced by phosphoenol pyruvate, and inhibited by ATP and AMP in a competitive manner.

Expression and characterization of NADPH-cytochrome P450 reductase from Trametes versicolor in Escherichia coli

Trametes versicolor has strong ability to degrade environmental organic pollutants. NADPH-cytochrome P450 reductase (CPR) of T. versicolor transfers electron to cytochrome P450s (CYPs) and participates in the degradation process of organic pollutants. Sequence analysis showed that the genome of T. versicolor contains 1 potential CPR and multiple potential CYP sequences. To further study the molecular mechanism for the involvement of T. versicolor CPR in the cellular degradation of organic pollutants, a CPR gene from T. versicolor was cloned and heterologously expressed in Escherichia coli. Subsequently, the main properties of the recombinant enzyme were investigated. A truncated CPR protein lacking the predicted membrane anchor region (residues 1-24), named CPRΔ24, was overexpressed as a soluble form in E. coli. The recombinant CPRΔ24 protein showed a molecular weight consistent with the theoretical value of 78 kDa. Recombinant CPRΔ24 was purified using a Ni²⁺-chelating column followed by size exclusion chromatography. The specific activity of the purified CPRΔ24 was 5.82 U/mg. The CPRΔ24 enzyme displayed the maximum activity at 35 ℃ and pH 8.0. It has different degrees of tolerance against several types of metal ions and organic solvents. The apparent Km and kcat values of recombinant CPRΔ24 for NADPH were 19.7 μmol/L and 3.31/s, respectively, and those for the substrate cytochrome c were 25.9 μmol/L and 10.2/s, respectively, under conditions of 35 ℃ and pH 8.0. The above research provides the basis for exploring the functional mechanism of T. versicolor CPR in the degradation pathway of environmental organic pollutants.

A novel gene, encoding 3-aminobenzoate 6-monooxygenase, involved in 3-aminobenzoate degradation in Comamonas sp. strain QT12.

The biodegradation pathway of 3-aminobenzoate has been documented, but little is known about the sequence and biochemical properties of the proteins involved. In the present study, a 10,083-bp DNA fragment involved in 3-aminobenzoate degradation was identified in 3-aminobenzoate-degrading Comamonas sp. strain QT12. The mabA gene, whose encoded protein shares 39% amino acid sequence identity with 3-hydroxybenzoate 6-hydroxylase of Polaromonas naphthalenivorans CJ2, was identified on this DNA fragment, and the mabA-disrupted mutant was unable to grow on and convert 3-aminobenzoate. MabA was heterologously expressed in Escherichia coli and purified to homogeneity as an approximately ~ 48-kDa His-tagged protein. It was characterized as 3-aminobenzoate 6-hydroxylase capable of catalyzing the conversion of 3-aminobenzoate to 5-aminosalicylate, incorporating one oxygen atom from dioxygen into the product. It contains a non-covalent but tightly bound FAD as the prosthetic group and NADH as an external electron donor. 5-Aminosalicylate was produced with equimolar consumption of NADH. The apparent Km and kcat values of the purified enzyme for 3-aminobenzoate were 158.51 ± 4.74μM and 6.49 ± 0.17s-1, respectively, and those for NADH were 189.85 ± 55.70μM and 7.41 ± 1.39s-1, respectively. The results suggest that mabA is essential for 3-aminobenzoate degradation in strain QT12, and that 3-aminobenzoate is the primary and physiological substrate of MabA.

Substrate specificity of healthy human sera IgG antibodies with peroxidase and oxydoreductase activities.

We have carried out an analysis of whether blood IgG antibodies can protect humans from oxidative stress by oxidizing different harmful compounds. A somewhat unexpected result was obtained. We show here for the first time that healthy human sera IgGs with the peroxidase (in the presence H2O2) efficiently oxidize different compounds: 3,3′-diaminobenzidine (1; DAB), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (2; ATBS), o-phenylenediamine (3; OPD), homovanillic acid (4; HVA), α-naphthol (5), 5-aminosalicylic acid (6; 5-ASA) and 3-amino-9-ethylcarbazole (7; AEC), but seven of nine IgG preparations from different volunteers cannot oxidize p-hydroquinone (8: pHQ). The average apparent kcat values in the H2O2-dependent oxidation by human IgGs decreased in the following order (min−1): ATBS (73.7) ≥ DAB (66.3) > AEC (38.0) ≥ HVA (19.8) ≥ α-naphthol (8.6) > OPD (0.62) ≥ 5-ASA (0.48) > pHQ (0.24). In the absence of H2O2 (oxidoreductase activity), the relative average kcat values decreased in the following order (min−1): DAB (52.1) ≥ ATBS (50.5) > OPD (0.25). The peroxidase average activity of human IgGs was higher than the oxidoreductase one: 1.2-, 1.5- and 2.5-fold for DAB, ATBS and OPD, respectively. It should be assumed that antibodies can oxidize in addition to the large number of other different compounds analysed by us. As a whole, the specific wide repertoire of polyclonal human IgGs oxidizing various compounds could play an important role in protecting humans from oxidative stress and serve as an additional natural system destroying H2O2 and different toxic mutagenic and carcinogenic compounds.

Cloning and expression of the carbaryl hydrolase gene mcbA and the identification of a key amino acid necessary for carbaryl hydrolysis

Carbamate hydrolase is the initial and key enzyme for degradation of carbamate pesticides. In the present study, we report the isolation of a carbaryl-degrading strain Pseudomonas sp. XWY-1, the cloning of its carbaryl hydrolase gene (mcbA) and the characterization of McbA. Strain XWY-1 was able to utilize carbaryl as a sole carbon source and degrade it using 1-naphthol as an intermediate. Transposon mutagenesis identified a mutant of XWY-1M that was unable to hydrolyze carbaryl. The transposon-disrupted gene mcbA was cloned by self-formed adaptor PCR, then expressed in Escherichia coli BL21(DE3) and purified. McbA was able to hydrolyze carbamate pesticides including carbaryl, isoprocarb, fenobucarb, carbofuran efficiently, while it hydrolyzed aldicarb, and propoxur poorly. The optimal pH of McbA was 7.0 and the optimal temperature was 40°C. The apparent Km and kcat values of McbA for carbaryl were 77.67±12.31μM and 2.12±0.10s−1, respectively. Three amino acid residues (His467, His477 and His504) in the predicted polymerase/histidinol phosphatase-like domain were shown to be closely related to the activity of McbA, with His504 being the most important, as a replacement of His504 led to the complete loss of activity. This is the first study to identify key amino acids in McbA.

Impact of Redox Modifications on ERK2 Substrate Phosphorylation

Extracellular regulated kinase 2 (ERK2), which is the terminal serine/threonine protein kinase in the Ras-Raf-MEK-ERK signaling axis, is involved in the regulation of many cellular processes, including cell proliferation, differentiation, survival, and cell cycle progression. Recently, ERK2 was shown to undergo reversible oxidation, inhibiting reactivity toward Elk1 substrate, following growth factor stimulation in a number of cell types (Keyes et al., FRBM, 2017). Interestingly, one of the residues exhibiting oxidation sensitivity (C159), is located within the D-recruitment site (DRS). Since the DRS is involved in binding a subset of ERK2 substrates, we compared the ability of wild-type ERK2 and ERK2(C159S) to phosphorylate a model DRS peptide substrate, Sub-D, following treatment with various concentrations of H2O2. Kinetic analysis demonstrated that, while both wild-type ERK2 and ERK2(C159S) exhibit similar apparent kcat and Km values for Sub-D in the absence of H2O2, treatment with H2O2 causes more significant changes in kcat,app and Km,app for Sub-D in wild-type compared with the C159S mutant of ERK2. Together, these data suggest that redox modification of ERK2 may alter its ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter ERK2’s activity toward some, but not all, of its downstream substrates. To explore this possibility further, we investigated the impact of redox modification on ERK2’s global substrate selectivity using functional protein microarrays. Consistent with our hypothesis, ERK2-mediated phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2-dependent changes in their phosphorylation status, indicating that C159 oxidation controls ERK activity by modulating substrate specificity. Together, these studies offer important insights into redox regulation of ERK2 and provide potential points of signal integration between kinase- and redox-dependent signaling pathways.

Purification and partial characterization of a low molecular fibrinolytic serine metalloprotease C142 from the culture supernatant of Bacillus subtilis C142

Novel serine metalloprotease-like enzyme, C142 was purified from the culture supernatant of Bacillus subtilis C142. The C142 was purified to homogeneity by a two-step procedure with a 20.7-fold increase in specific activity and 0.9% recovery. The molecular mass of C142 was approximately 23.5kDa based on SDS-PAGE. The N-terminal amino acid sequence of the first 21 amino acids of C142 was AQSVPYGISQIKAPALHSQGY. Its optimum pH, optimum temperature, pH stability, and thermal stability were pH 6, 40°C, pH 6–8, and 20–35°C, respectively. C142 was strongly inhibited by PMSF and EGTA, suggesting that C142 was a serine metalloprotease-like enzyme. C142 showed the highest specificity toward the substrate for t-PA. The apparent Km, Vmax, and Kcat values of C142 toward H-d-Ile-Pro-Arg-pNA were determined as 0.34mM, 0.25mmolmg−1min−1, and 46.83s−1. C142 exhibited fibrinolytic activity, which is stronger than that of plasmin. C142 hydrolyzed Aα, and Bβ-chains of fibrinogen, but did not cleave γ-chains. C142 had antithrombotic effect in three animal models. C142 was devoid of hemorrhagic activity at a dose of 20,000FU/kg. Taken together, our results indicate that B. subtilis C142 produces a serine metalloprotease-like enzyme/fibrinolytic enzyme and this enzyme might be used as a new thrombolytic agent.

Cloning and Characterization of Filamentous Fungal S-Nitrosoglutathione Reductase from Aspergillus nidulans.

S-Nitrosoglutathione reductase (GSNOR) metabolizes S-nitrosoglutathione (GSNO) and has been shown to play important roles in regulating cellular signaling and formulating host defense by modulating intracellular nitric oxide levels. The enzyme has been found in bacterial, yeast, mushroom, plant, and mammalian cells. However, to date, there is still no evidence of its occurrence in filamentous fungi. In this study, we cloned and investigated a GSNOR-like enzyme from the filamentous fungus Aspergillus nidulans. The enzyme occurred in native form as a homodimer and exhibited low thermal stability. GSNO was an ideal substrate for the enzyme. The apparent Km and kcat values were 0.55 mM and 34,100 min(-1), respectively. Substrate binding sites and catalytic center amino acid residues based on those from known GSNORs were conserved in this enzyme, and the corresponding roles were verified using site-directed mutagenesis. Therefore, we demonstrated the presence of GSNOR in a filamentous fungus for the first time.