A Tropical Composting Operation Unit at São Paulo Zoo as a Source of Bacterial Proteolytic Enzymes.

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the São Paulo Zoo Park (SPZPF), São Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.

Functional roles of C-terminal extension (CTE) of salt-dependent peptidase activity of the Natrialba magadii extracellular protease (NEP).

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon Natrialba magadii. Many extracellular proteases have been characterized from archaea to bacteria as adapted to hypersaline environments retaining function and stability until 4.0M NaCl. As observed in other secreted halolysins, this stability can be related to the presence of a C-terminal extension (CTE) sequence. In the present work, we compared the biochemical properties of recombinant Nep protease with the truncated form at the 134 amino acids CTE (Nep∆CTE), that was more active in 4M NaCl than the non-truncated wild type enzyme. Comparable to the wild type, Nep∆CTE protease is irreversibly inactivated at low salt solutions. The substrate specificity of the truncated Nep∆CTE was similar to that of wild type form as demonstrated by a combinatorial library of FRET substrates. The enzyme stability, the effect of different salts and the thermodynamics assays using different lengths of substrates demonstrated similarities between the two forms. Altogether, these data provide further information on the stability and structural determinants of halolysins under different salinities, especially concerning the enzymatic behavior.

Nuclear matrix metalloproteinase-2 in the cardiomyocyte and the ischemic-reperfused heart.

Matrix metalloproteinases (MMPs) are zinc-dependent proteases involved in intra- and extra-cellular matrix remodeling resulting from oxidative stress injury to the heart. MMP-2 was the first MMP to be localized to the nucleus; however, its biological functions there are unclear. We hypothesized that MMP-2 is present in the nucleus under normal physiological conditions but increases during myocardial ischemia-reperfusion (I/R) injury-induced oxidative stress, proteolyzing nuclear structural proteins. Lamins are intermediate filament proteins that provide structural support to the nucleus and are putative targets of MMP-2. To identify lamin susceptibility to MMP-2 proteolysis, purified lamin A or B was incubated with MMP-2 in vitro. Lamin A, but not lamin B, was proteolysed by MMP-2 into an approximately 50kDa fragment, which was also predicted by in silico cleavage site analysis. Immunofluorescent confocal microscopy and subcellular fractionation showed MMP-2 both in the cytosol and nuclei of neonatal rat ventricular myocytes. Rat hearts were isolated and perfused by the Langendorff method aerobically, or subjected to I/R injury in the presence or absence of o-phenanthroline, an MMP inhibitor. Nuclear fractions extracted from I/R hearts showed increased MMP-2 activity, but not protein level. The level of troponin I, a known sarcomeric target of MMP-2, was rescued in I/R hearts treated with o-phenanthroline, demonstrating the efficacy of MMP inhibition. However, lamin A or B levels remained unchanged in I/R hearts. MMP-2 has a widespread subcellular distribution in cardiomyocytes, including a significant presence in the nucleus. The increase in nuclear MMP-2 activity seen during stunning injury here, indicates yet unknown biological actions, other than lamin proteolysis, which may require more severe ischemia to effect.

The natural flavone fukugetin as a mixed-type inhibitor for human tissue kallikreins.

The human tissue kallikreins (KLK1-KLK15) comprise a family of 15 serine peptidases detected in almost every tissue of the human body and that actively participate in many physiological and pathological events. Some kallikreins are involved in diseases for which no effective therapy is available, as for example, epithelial disorders, bacterial infections and in certain cancers metastatic processes. In recent years our group have made efforts to find inhibitors for all kallikreins, based on natural products and synthetic molecules, and all the inhibitors developed by our group presented a competitive mechanism of inhibition. Here we describe fukugetin, a natural product that presents a mixed-type mechanism of inhibition against KLK1 and KLK2. This type of inhibitor is gaining importance today, especially for the development of exosite-type inhibitors, which present potential to selectively inhibit the enzyme activity only against specific substrate.

Analysis of catalytic properties of tripeptidyl peptidase I (TTP-I), a serine carboxyl lysosomal protease, and its detection in tissue extracts using selective FRET peptide substrate.

Tripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endo- peptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559-72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca(2+) and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at F-F bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher k(cat)/K(M) compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys.

Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts.

Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications.

Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts

Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications.

The Serine Protease Pic From Enteroaggregative Escherichia coli Mediates Immune Evasion by the Direct Cleavage of Complement Proteins.

Enteroaggregative and uropathogenic Escherichia coli, Shigella flexneri 2a, and the hybrid enteroaggregative/Shiga toxin-producing E. coli strain (O104:H4) are important pathogens responsible for intestinal and urinary tract infections, as well as sepsis and hemolytic uremic syndrome. They have in common the production of a serine protease called Pic. Several biological roles for Pic have been described, including protection of E. coli DH5α from complement-mediated killing. Hereby we showed that Pic significantly reduces complement activation by all 3 pathways. Pic cleaves purified C3/C3b and other proteins from the classic and lectin pathways, such as C4 and C2. Cleavage fragments of C3, C4, and C2 were also observed with HB101(pPic1) culture supernatants, and C3 cleavage sites were mapped by fluorescence resonance energy transfer peptides. Experiments using human serum as a source of complement proteins confirmed Pic proteolytic activity on these proteins. Furthermore, Pic works synergistically with the human complement regulators factor I and factor H, promoting inactivation of C3b. In the presence of both regulators, further degradation of C3 α' chain was observed. Therefore, Pic may contribute to immune evasion of E. coli and S. flexneri, favoring invasiveness and increasing the severity of the disorders caused by these pathogens.

Specificity studies on Kallikrein-related peptidase 7 (KLK7) and effects of osmolytes and glycosaminoglycans on its peptidase activity.

KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F, Y or M, and its S1' and S2' subsites showed selectivity for hydrophilic amino acids, particularly R and K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz-MISLM(↓)KRPPGFSPF(↓)RSSRI-NH2 ((↓)indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-NLY(↓)RVE-Q-EDDnp is the best synthetic substrate so far described for KLK7 [kcat/Km=455 (mMs)(-1)] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (Ki=33mM) and is present in skin moisturizing factor (124mM). The KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed.

Analysis of peptidase activities of a cathepsin B-like (TcoCBc1) from Trypanosoma congolense.

The substrate specificity of TcoCBc1 was evaluated using two internally quenched fluorescent peptide libraries with randomized sequences designed to detect carboxydipeptidase (Abz-GXXZXK(Dnp)-OH) and endopeptidase (Abz-GXXZXXQ-EDDnp) activities at acidic and neutral pHs, respectively. All the data obtained with TcoCBc1 were compared with those of human cathepsin B, including the pH profiles of the hydrolytic reactions. The most relevant observation is the preference of TcoCBc1 for substrates with a pair of acidic amino acids at positions P(2) and P(1) for its carboxydipeptidase activity and the well acceptance for E and D at P(1) position for endopeptidase activity. These peculiar preferences for negatively charged groups of TcoCBc1 and its requirements for carboxydipeptidase activity were also observed on Abz labeled analogues of bradykinin (Abz-RPPG(↓)FSAFR-OH, Abz-RPPG(↓)FS(↓)AF-OH, Abz-RPPG(↓)DE(↓)AF-OH) and angiotensin I (Abz-DR(↓)VYIHAFHL-OH), where (↓) indicates the cleavage site. TcoCBc1 was modeled based on the atomic coordinates of the cathepsin B from Trypanosoma brucei and the positively charged environment in TcoCBc1 catalytic site contrasts with the negatively charged environment in human cathepsin B. The preferences of S1 and S2 subsites of TcoCBc1 for acidic amino acids have to be taken into consideration for future studies of physiological roles of TcoCBc1 as for instance in apoptotic processes of Trypanosoma congolense.

Enzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptors.

Gyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. The serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. In the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. The kcat/KM values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. For the sh and lg PAR-2 mimetic peptides the kcat/KM values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. The kcat/KM values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-1, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or by synthetic PAR-1 agonist in cultured astrocytes. Taken together, the data presented here allow us showing that gyroxin cleaves PARs-mimetic peptides slowly and it does not induce activation of PARs in astrocytes. Although gyroxin does not mobilize calcium it was shown to interfere with PARs activation by thrombin and PAR-1 agonist. The determination of gyroxin enzymatic specificity and kinetics on PAR-1, -2, -3, and -4 will potentially help to fill the gap in the knowledge in this field, as the PARs are still believed to have a key role for the gyroxin biological effects.

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: insights into substrate selectivity and kinetic behavior.

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.

Mycoplasma hyopneumoniae in vitro peptidase activities: identification and cleavage of kallikrein-kinin system-like substrates.

Bacterial proteases are important for metabolic processes and pathogenesis in host organisms. The bacterial swine pathogen Mycoplasma hyopneumoniae has 15 putative protease-encoding genes annotated, but none of them have been functionally characterized. To identify and characterize peptidases that could be relevant for infection of swine hosts, we investigated the peptidase activity present in the pathogenic 7448 strain of M. hyopneumoniae. Combinatorial libraries of fluorescence resonance energy transfer peptides, specific inhibitors and pH profiling were used to screen and characterize endopeptidase, aminopeptidase and carboxypeptidase activities in cell lysates. One metalloendopeptidase, one serine endopeptidase, and one aminopeptidase were detected. The detected metalloendopeptidase activity, prominent at neutral and basic pH ranges, was due to a thimet oligopeptidase family member (M3 family), likely an oligoendopeptidase F (PepF), which cleaved the peptide Abz-GFSPFRQ-EDDnp at the F-S bond. A chymotrypsin-like serine endopeptidase activity, possibly a subtilisin-like serine protease, was prominent at higher pH levels, and was characterized by its preference for a Phe residue at the P1 position of the substrate. The aminopeptidase P (APP) activity showed a similar profile to that of human membrane-bound APP. Genes coding for these three peptidases were identified and their transcription was confirmed in the 7448 strain. Furthermore, M. hyopneumoniae cell lysate peptidases showed effects on kallikrein-kinin system-like substrates, such as bradykinin-derived substrates and human high molecular weight kininogen. The M. hyopneumoniae peptidase activities, here characterized for the first time, may be important for bacterial survival strategies and thus represent possible targets for drug development against M. hyopneumoniae swine infections.

Isomannide derivatives as new class of inhibitors for human kallikrein 7.

Human kallikrein 7 (KLK7) is a potential target for the treatment of skin inflammation and cancer. Despite its potential, few KLK7-specific small-molecule inhibitors have been reported in the literature. As an extension of our program to design serine protease inhibitors, here we describe the in vitro assays and the investigation of the binding mechanism by molecular dynamics simulation of a novel class of pseudo-peptide inhibitors derived from isomannide. Of the inhibitors tested, two inhibited KLK7 with K(i) values in the low micromolar range (9g=1.8µM; 9j=3.0µM). Eadie-Hofstee and Dixon plots were used to evaluate the competitive mechanism of inhibition for the molecules. Calculated binding free energies using molecular MM/PB(GB)SA approach are in good agreement with experimental results, suggesting that the inhibitors share the same binding mode, which is stabilized by hydrophobic interactions and by a conserved network of hydrogen bonds. The promising results obtained in this study make these compounds valid leads for further optimization studies aiming to improve the potency of this new class of kallikrein inhibitors.

Kinetic characterization of gyroxin, a serine protease from Crotalus durissus terrificus venom.

This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 °C to 45 °C, and increases of NaCl concentration up to 1 M led to activity decreases. The preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity.

Correlation between catalysis and tertiary structure arrangement in an archaeal halophilic subtilase.

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M (NaCl), Nep behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3-1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.

Internally quenched fluorescent peptide libraries with randomized sequences designed to detect endopeptidases.

Identification of synthetic peptide substrates for novel peptidases is an essential step for their study. With this purpose we synthesized fluorescence resonance energy transfer (FRET) peptide libraries Abz (or MCA)-GXXXXXQ-EDDnp and Abz (or MCA)-GXXZXXQ-EDDnp, where X consists of an equimolar mixture of all amino acids, the Z position is fixed with one of the proteinogenic amino acids (cysteine was excluded), Abz (ortho-aminobenzoic acid) or MCA ([7-amino-4-methyl]coumarin) is the fluorescence donor and Q-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine]) is the fluorescence acceptor. The peptide libraries MCA-GXXX↓XXQ-EDDnp and MCA-GXXZ↓XXQ-EDDnp were cleaved as indicated (↓) by trypsin, chymotrypsin, cathepsin L, pepsin A, and Eqolisin as confirmed by Edman degradation of the products derived from the digestion of these libraries. The best hydrolyzed Abz-GXXZXXQ-EDDnp sublibraries by these proteases, including Dengue 2 virus NS2B-NS3 protease, contained amino acids at the Z position that are reported to be well accepted by their S(1) subsite. The pH profiles of the hydrolytic activities of these canonical proteases on the libraries were similar to those reported for typical substrates. The FRET peptide libraries provide an efficient and simple approach for detecting nanomolar concentrations of endopeptidases and are useful for initial specificity characterization as performed for two proteases secreted by a Bacillus subtilis.

Biological evaluation and docking studies of natural isocoumarins as inhibitors for human kallikrein 5 and 7.

Human kallikrein 5 and 7 (KLK5 and KLK7) are trypsin-like and chymotrypsin-like serine proteases, respectively, and promising targets for the treatment of skin desquamation, inflammation and cancer. In an effort to develop new inhibitors for these enzymes, we carried out enzymatic inhibition assays and docking studies with three isocoumarin compounds. Some promising inhibitors were uncovered, with vioxanthin and 8,8'-paepalantine being the most potent competitive inhibitors of KLK5 (K(i)=22.9 µM) and KLK7 (K(i)=12.2 µM), respectively. Our docking studies showed a good correlation with the experimental results, and revealed a distinct binding mode for the inhibitors at the binding sites of KLK5 and KLK7. In addition, the docking results suggested that the formation of hydrogen bonds at the oxyanion hole is essential for a good inhibitor.

Yellow fever virus NS2B/NS3 protease: hydrolytic properties and substrate specificity.

Here we report the hydrolytic behavior of recombinant YFV NS2B/NS3 protease against FRET substrates mimicking the prime and non-prime region of the natural polyprotein cleavage sites. While the P2-P'1 motif is the main factor associated with the catalytic efficiency of Dengue (DV) and West Nile Virus (WNV) protease, we show that the k(cat)/K(m) of YFV NS2B/NS3 varied by more than two orders of magnitude, despite the presence of the same motif in all natural substrates. The catalytic significance of this homogeneity - a unique feature among worldwide prominent flavivirus - was kinetically analyzed using FRET peptides containing all possible combinations of two and three basic amino acids in tandem, and Arg and Lys residues produced distinct effects on k(cat)/K(m). The parallel of our data with those obtained in vivo by Chambers et al. (1991) restrains the idea that these sites co-evolved with the NS2B/NS3 protease to promote highly efficient hydrolysis and supports the notion that secondary substrate interaction distant from cleavage sites are the main factor associated with the different hydrolytic rates on YFV NS2B-NS3pro natural substrates.

Increase of SARS-CoV 3CL peptidase activity due to macromolecular crowding effects in the milieu composition.

The 3C-like peptidase of the severe acute respiratory syndrome virus (SARS-CoV) is strictly required for viral replication, thus being a potential target for the development of antiviral agents. In contrast to monomeric picornavirus 3C peptidases, SARS-CoV 3CLpro exists in equilibrium between the monomer and dimer forms in solution, and only the dimer is proteolytically active in dilute buffer solutions. In this study, the increase of SARS-CoV 3CLpro peptidase activity in presence of kosmotropic salts and crowding agents is described. The activation followed the Hofmeister series of anions, with two orders of magnitude enhancement in the presence of Na2SO4, whereas the crowding agents polyethylene glycol and bovine serum albumin increased the hydrolytic rate up to 3 times. Kinetic determinations of the monomer dimer dissociation constant (K(d)) indicated that activation was a result of a more active dimer, without significant changes in K(d) values. The activation was found to be independent of substrate length and was derived from both k(cat) increase and K(m) decrease. The viral peptidase activation described here could be related to the crowded intracellular environment and indicates a further fine-tuning mechanism for biological control, particularly in the microenvironment of the vesicles that are induced in host cells during positive strand RNA virus infection.