Improved catalytic efficiency of catalase from Bacillus subtilis by rational mutation of Lys114

Alkaline catalases with good properties are desirable in the textile industry. In the present work, by applying the PoPMuSiC algorithm for the calculation of the folding free energy, Lys114 of a Bacillus catalase was rationally selected and engineered to improve the thermostability. Interestingly, the Lys114Tyr, Lys114Val, Lys114Met and Lys114Ile variants showed higher catalytic efficiencies when compared with the wild-type protein. In particular, the Lys114Tyr variant showed the highest catalytic efficiency, which was 5.3-fold of the wild-type catalase. The production of the Lys114Tyr variant may represent an improved catalase suitable for industrial purposes.     

Characterization of Native and Recombinant Forms of an Unusual Cobalt-Dependent Proline Dipeptidase (Prolidase) from the Hyperthermophilic Archaeon Pyrococcus furiosus

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co²⁺ ions (K_d, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co²⁺ could be replaced by Mn²⁺ (resulting in a 25% decrease in activity) but not by Mg²⁺, Ca²⁺, Fe²⁺, Zn²⁺, Cu²⁺, or Ni²⁺. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100°C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the K_m values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater V_max value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.     

Engineering a Disulfide Bond in the Lid Hinge Region of Rhizopus chinensis Lipase: Increased Thermostability and Altered Acyl Chain Length Specificity

The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for “interfacial activation” is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the “Disulfide by Design” algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t _1/2 value at 60°C and a 7°C increase of T _m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k _cat) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.     

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

High thermostability is required for alkaline α-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline α-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants—P35C-G426C, G116C-Q120C, and R436C-M480C—of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (k_cat/K_m) increased from 1.8 × 10⁴ to 2.4 × 10⁴ liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.     

Identification and Characterization of d-Hydroxyproline Dehydrogenase and Δ1-Pyrroline-4-hydroxy-2-carboxylate Deaminase Involved in Novel l-Hydroxyproline Metabolism of Bacteria: METABOLIC CONVERGENT EVOLUTION*

l-Hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert l-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, d-hydroxyproline dehydrogenase and Δ¹-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. d-Hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (d-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α₄β₄γ₄), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the l-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on l-hydroxyproline (as well as d-hydroxyproline) but not l- and d-proline, indicating that this pathway is related only to l-hydroxyproline degradation, which is not linked to proline metabolism.     

Structure–Function Study of Recombinant Onconase Variants

A method for expression of an onconase gene leading to a soluble form of the protein was developed. The enzymatic and cytotoxic properties of the protein's recombinant forms were studied. Recombinant onconase with an additional N-terminal Met residue isolated in non-denaturing conditions did not substantially differ from the native enzyme in ribonucleolytic activity. The addition of a 33-mer peptide containing auxiliary elements for the simplification of isolation and detection of the recombinant protein did not affect the enzyme properties of onconase. The method proposed is useful for the onconase structure–function relation studies and enables construction of onconase-based fusion proteins for anticancer therapy.     

Production and Characterization of N- and C-terminally Truncated Mtx2: a Mosquitocidal Toxin from Bacillus sphaericus

Mosquitocidal toxin 2 (Mtx2) is a mosquito-larvicidal protein produced during vegetative stage of Bacillus sphaericus. The toxin consists of 292 amino acids and has a molecular weight of 31.8 kDa. To determine the active core region of the toxin, amino acids at N- and C-termini were sequentially removed. Deletion up to 23 amino acids from the N-terminus (Met1-Tyr23) did not significantly affect protein production and the toxin activity, whereas removal of 26 amino acids from the N-terminus (Met1-Lys26) completely abolished toxicity even though the protein production remained unchanged. Deletion of only 5 amino acids from the C-terminal end yielded the protein that could not be solubilized and rendered the toxin inactive. The results demonstrated that the C-terminal end of Mtx2 is required for proper folding and toxicity. Amino acids at the N-terminus up to Tyr23 did not play a significant role in protein production and toxicity whereas amino acids between Thr24 and Lys26 are required for full toxicity.     

Improvement of catalytic efficiency and thermostability of recombinant Streptomyces griseus trypsin by introducing artificial peptide

Streptomyces trypsin is one of the serine proteinases in Streptomyces griseus and acts as a key mediator during cell growth and differentiation. S. griseus trypsin (SGT) could be successfully expressed in Pichia pastoris by engineering the natural propeptide APNP. In this study, the recombinant Exmt with peptide YVEF and the wild-type SGT were comparatively investigated in detail. The recombinant Exmt showed significantly increased thermostability which t _1/2 value was 3.89-fold of that of the SGT at 40 °C. Moreover, the catalytic efficiency (referring to the specificity constant, k _cat/K _m) and pH tolerance of Exmt were also improved. In silico modeling analysis uncovered that introduction of the peptide YVEF resulted in a broadened substrate binding pocket and closer catalytic triad (His⁵⁷, Asp¹⁰² and Ser¹⁹⁵). The intramolecular Hydrogen bonds and the cation π-interactions were also dramatically increased. The results indicated that engineering of the N-terminus with artificial peptides might be an effective approach for optimizing the properties of the target enzymes.     

A novel thermostable arylesterase from the archaeon Sulfolobus solfataricus P1: purification, characterization, and expression

A novel thermostable arylesterase, a 35-kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 94°C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90°C. In addition, the purified enzyme showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity besides showing an arylesterase activity toward aromatic esters: it exhibits not only carboxylesterase activity toward tributyrin and p-nitrophenyl esters containing unsubstituted fatty acids from butyrate (C₄) to palmitate (C₁₆), but also paraoxonase activity toward organophosphates such as p-nitrophenylphosphate, paraoxon, and methylparaoxon. The k_cat/K_m ratios of the enzyme for phenyl acetate and paraoxon, the two most preferable substrates among all tested, were 30.6 and 119.4 s⁻¹·μM⁻¹, respectively. The arylesterase gene consists of 918 bp corresponding to 306 amino acid residues. The deduced amino acid sequence shares 34% identity with that of arylesterase from Acinetobacter sp. strain ADP1. Furthermore, we successfully expressed active recombinant S. solfataricus arylesterase in Escherichia coli. Together, our results show that the enzyme is a serine esterase belonging to the A-esterases and contains a catalytic triad composed of Ser156, Asp251, and His281 in the active site.     

Entry into cells and selective degradation of tRNAs by a cytotoxic member of the RNase A family

Onconase (P-30 protein), an enzyme in the ribonuclease A superfamily, exerts cytostatic, cytotoxic, and antiviral activity when added to the medium of growing mammalian cells. We find that onconase enters living mammalian cells and selectively cleaves tRNA with no detectable degradation of rRNA. The RNA specificity of onconase in vitro using reticulocyte lysate and purified RNA substrates indicates that proteins associated with rRNA protect the rRNA from the onconase ribonucleolytic action contributing to the cellular tRNA selectivity of onconase. The onconase-mediated tRNA degradation in cells appears to be accompanied by increased levels of tRNA turnover and induction of tRNA synthesis perhaps in response to the selective toxin-induced loss of tRNA. Degradation products of tRNA(3)(Lys), which acts as a primer for HIV-1 replication, were clearly detected in cells infected with HIV-1 and treated with sublethal concentrations of onconase. However, a new synthesis of tRNA(3)(Lys) also seemed to occur in these cells resulting in plateauing of the steady-state levels of this tRNA. We conclude that the degradation of tRNAs may be a primary factor in the cytotoxic activity of onconase.     

Synergism between Basic Asp49 and Lys49 Phospholipase A2 Myotoxins of Viperid Snake Venom In Vitro and In Vivo

Two subtypes of phospholipases A₂ (PLA₂s) with the ability to induce myonecrosis, ‘Asp49’ and ‘Lys49’ myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca²⁺ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 myotoxin as low as 0.1% of the Lys49 myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 myotoxin. Asp49 myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 myotoxin did not. In contrast to myotoxic Asp49 PLA₂, an acidic non-toxic PLA₂ from the same venom did not markedly synergize with Lys49 myotoxin, revealing a functional difference between basic and acidic PLA₂ enzymes. It is suggested that Asp49 myotoxins synergize with Lys49 myotoxins by virtue of their PLA₂ activity. In addition to the membrane-destabilizing effect of this activity, Asp49 myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA₂ myotoxins acting synergistically in viperid venoms.     

Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3" exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3" pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5" phosphate of the 3" fragment, which is not required for U removal. The ability of the 3" U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3" exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (R_p)α-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.     

Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions

Improving enzyme thermostability is of importance for widening the spectrum of application of enzymes. In this study, a structure-based rational design approach was used to improve the thermostability of a highly active, wide-pH-range-adaptable, and stable endopolygalacturonase (PG8fn) from Achaetomium sp. strain Xz8 via the optimization of charge-charge interactions. By using the enzyme thermal stability system (ETSS), two residues—D244 and D299—were inferred to be crucial contributors to thermostability. Single (D244A and D299R) and double (D244A/D299R) mutants were then generated and compared with the wild type. All mutants showed improved thermal properties, in the order D244A < D299R < D244A/D299R. In comparison with PG8fn, D244A/D299R showed the most pronounced shifts in temperature of maximum enzymatic activity (T_max), temperature at which 50% of the maximal activity of an enzyme is retained (T₅₀), and melting temperature (T_m), of about 10, 17, and 10.2°C upward, respectively, with the half-life (t_1/2) extended by 8.4 h at 50°C and 45 min at 55°C. Another distinguishing characteristic of the D244A/D299R mutant was its catalytic activity, which was comparable to that of the wild type (23,000 ± 130 U/mg versus 28,000 ± 293 U/mg); on the other hand, it showed more residual activity (8,400 ± 83 U/mg versus 1,400 ± 57 U/mg) after the feed pelleting process (80°C and 30 min). Molecular dynamics (MD) simulation studies indicated that mutations at sites D244 and D299 lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity. This study reveals the importance of charge-charge interactions in protein conformation and provides a viable strategy for enhancing protein stability.     

The effect of different dietary ratios of lysine,arginine and methionine on protein nitration and oxidation reactions in turkey tissues and DNA

An assumption was made in the study that the optimal inclusion levels and ratios of lysine (Lys), arginine (Arg) and methionine (Met) in diets with Lys content consistent with National Research Council (NRC) recommendations (1994) contribute to stimulate the antioxidant defense system and prevent disorders resulting from the oxidation and nitration of biologically important molecules. The experiment was carried out on 864 one-day-old Hybrid Converter turkeys divided into six experimental groups (8 replicates per group and 18 birds per replicate) receiving different levels of Arg and Met. Chickens from group Arg₉₀Met₃₀ received 90% Arg and 30% Met relative to Lys; Arg₉₀Met₄₅ – 90% Arg and 45% Met relative to Lys; Arg₁₀₀Met₃₀ – 100% Arg and 30% Met relative to Lys; Arg₁₀₀Met₄₅ – 100% Arg and 45% Met relative to Lys; Arg₁₁₀Met₃₀ – 110% Arg and 30% Met relative to Lys and Arg₁₁₀Met₄₅ – 110% Arg level and 45% Met level relative to the content of dietary Lys. In comparison with turkeys fed diets with moderate Arg content (100% of Lys content), a decrease in dietary Arg level (90% of Lys content) led to a decrease in plasma 3-nitrotyrosine (3-NT) concentration (163.6 vs. 141.0), whereas an increase in dietary Arg level (110% of Lys content) led to an increase in plasma 3-NT concentration (163.6 vs. 202.6). In comparison with turkeys fed diets with moderate Arg content (100% of Lys content), the lowest dietary Arg level (90% of Lys content) decreased superoxide dismutase (SOD) activity in the intestinal wall (19.68 vs. 17.41) and in the liver (11.51 vs. 7.94), increased SOD activity in the blood (507.6 vs. 961.4) and in breast muscles (6.26 vs. 7.43) and increased the concentration of malondiadehyde in breast muscles (1.10 vs. 1.50). An increase in dietary Met content from 30 to 45% of Lys content caused a decrease in plasma protein carbonyl concentration (4.33 vs. 3.8) and catalase activity in breast muscles (54.70 vs. 49.66), and an increase in SOD activity in the liver (8.90 vs. 10.41). The highest dietary Arg level (110% of Lys content) did not induce the oxidation of lipids, proteins or DNA, but it increased the risk of protein nitration. The lowest dietary Arg level (90% of Lys content) deteriorated the antioxidant status of turkeys. Regardless of dietary Arg levels, an increase in Met content from 30 to 45% of Lys content stimulated the antioxidant defense system of turkeys.     

Improvement of the Thermostability and Activity of a Pectate Lyase by Single Amino Acid Substitutions, Using a Strategy Based on Melting-Temperature-Guided Sequence Alignment

In the vast number of random mutagenesis experiments that have targeted protein thermostability, single amino acid substitutions that increase the apparent melting temperature (T_m) of the enzyme more than 1 to 2°C are rare and often require the creation of a large library of mutated genes. Here we present a case where a single beneficial mutation (R236F) of a hemp fiber-processing pectate lyase of Xanthomonas campestris origin (PL_Xc) produced a 6°C increase in T_m and a 23-fold increase in the half-life at 45°C without compromising the enzyme's catalytic efficiency. This success was based on a variation of sequence alignment strategy where a mesophilic amino acid sequence is matched with the sequences of its thermophilic counterparts that have established T_m values. Altogether, two-thirds of the nine targeted single amino acid substitutions were found to have effects either on the thermostability or on the catalytic activity of the enzyme, evidence of a high success rate of mutation without the creation of a large gene library and subsequent screening of clones. Combination of R236F with another beneficial mutation (A31G) resulted in at least a twofold increase in specific activity while preserving the improved T_m value. To understand the structural basis for the increased thermal stability or activity, the variant R236F and A31G R236F proteins and wild-type PL_Xc were purified and crystallized. By structure analysis and computational methods, hydrophobic desolvation was found to be the driving force for the increased stability with R236F.     

Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs

The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2′O-methyladenosine (A_m), generating N6, 2′O-dimethyladenosine (m⁶A_m) when A_m is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and A_m), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2′O-methylated A_m over unmodified A nucleosides is due mainly to increased binding affinity for A_m. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5′-A_m oligonucleotide with approximately the same affinity as that of a cap analog (K_M = 0.4 versus 0.3 μM). In addition, PCIF1 methylates the uncapped 5′-A_m with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs.     

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO₂. They are activated by Mn²⁺, a metal ion that coordinates to the protein through the ?-amino group of a lysine residue, the N_?-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ?-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ?-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn²⁺ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn²⁺ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn²⁺. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.     

Different selectivities of oxidants during oxidation of methionine residues in the α-1-proteinase inhibitor

Oxidation of the reactive site methionine (Met) in α-1-proteinase inhibitor (α-1-PI) to methionine sulfoxide (Met(O)) is known to cause depletion of its elastase inhibitory activity. To estimate the selectivity of different oxidants in converting Met to Met(O) in α-1-PI, we measured the molar ratio Met(O)/α-1-PI at total inactivation. This ratio was determined to be 1.2 for both the myeloperoxidase/H₂O₂/chloride system and the related compound NH₂Cl. With taurine monochloramine, another myeloperoxidase-related oxidant, 1.05 mol Met(O) were generated per mol α-1-PI during inactivation. These oxidants attack preferentially one Met residue in α-1-PI, which is identical with Met 358, as concluded from the parallelism of loss of elastase inhibitory activity and oxidation of Met. A similar high specificity for Met oxidation was determined for the xanthine oxidase-derived oxidants. In contrast, the ratio found for ozone and m-chloroperoxybenzoic acid was 6.0 and 5.0, respectively, indicating oxidation of additional Met residues besides the reactive site Met in α-1-PI, i.e. unselective action of these oxidants. Further studies were performed on the efficiency of oxidants for total depletion of the elastase inhibitory capacity of α-1-PI. Ozone and m-chloroperoxybenzoic acid were 10-fold less effective and the superoxide anion/hydroxyl radicals were 30–50-fold less effective to inactivate the elastase inhibitory activity as compared to the myeloperoxidase-derived oxidants. The myeloperoxidase-related oxidants are discussed as important regulators of α-1-PI activity in vivo.     

Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity

The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X₃X₂X₁-aminomethylcoumarin) and by determining specificity constants (k_cat/K_m), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X₁ position, basic residues or Trp in the X₂ position, and Pro ≫ Ala > Trp in the X₃ position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.