RESUMEN
Ste24 enzymes, a family of eukaryotic integral membrane proteins, are zinc metalloproteases (ZMPs) originally characterized as "CAAX proteases" targeting prenylated substrates, including a-factor mating pheromone in yeast and prelamin A in humans. Recently, Ste24 was shown to also cleave nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders. Ste24 possesses a unique "α-barrel" structure consisting of seven transmembrane (TM) α-helices encircling a large intramembranous cavity (~14 000 Å3 ). The catalytic zinc, coordinated via a HExxH E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long-studied class of soluble ZMPs, and as a novel cavity-containing integral membrane protein protease has been minimally explored to date. Informed by homology to well-characterized soluble, gluzincin ZMPs, we develop a model of Ste24 that provides a conceptual framework for this enzyme family, suitable for development and interpretation of structure/function studies. The model consists of an interfacial, zinc-containing "ZMP Core" module surrounded by a "ZMP Accessory" module, both capped by a TM helical "α-barrel" module of as yet unknown function. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 absolutely conserved residues, apportioned unequally among the ZMP Core (18), ZMP Accessory (13), and α-barrel (7) modules. This Tripartite Architecture representation of Ste24 provides a unified image of this enzyme family.
Asunto(s)
Proteínas de la Membrana/química , Metaloendopeptidasas/química , Neprilisina/química , Termolisina/química , Secuencia de Aminoácidos , Bacillus/química , Bacillus/enzimología , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Geobacter/química , Geobacter/enzimología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Neprilisina/genética , Neprilisina/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Saccharomyces/química , Saccharomyces/enzimología , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Termolisina/genética , Termolisina/metabolismoRESUMEN
Solvent-exposed acidic/amide residue (Asp/Glu or Asn/Gln) exerts great effects on the thermostability of protein; however, experimental attempts appear to be time-consuming, so a more scientific, simple, and effective rational strategy is necessary. In this study, molecular dynamic (MD) simulation was performed to analyze two surface acidic residues (Asp37 and Glu119) of thermolysin (TLN) in mediating its thermostability. Root-mean-square-deviation (RMSD) was calculated to evaluate the thermosensitivity effect by acidic/amide substitutions. The wild-type TLN and three mutants (TLM1, TLM2, and TLM) presented significantly different thermostability effect. Four profiles of RMSD values demonstrated that the thermal insensitivity of variants were TLM2 > TLM > TLN > TLM1. As expected, the thermostability and half-life (at 60 °C) behavior of enzyme variants showed the same trends with the computational predictions, and it was worth noting that the half-life of TLM2 showed 3.1-fold longer than that of wild-type. The T m and T 50 of TLM2 were 9 and 7 °C higher, respectively, than that of wild-type enzyme. Rational substitution of acidic/amide residue in regulation of thermostability using MD simulation would be an efficient approach for instructional design to improve the thermostability.
Asunto(s)
Ácidos/química , Termolisina/metabolismo , Estabilidad de Enzimas , Cinética , Simulación de Dinámica Molecular , Mutagénesis , Desplegamiento Proteico , Temperatura , Termolisina/genéticaRESUMEN
Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of ß-rich N-terminal (residues 1-157) and α-rich C-terminal (residues 158-316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50-100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site.
Asunto(s)
Escherichia coli/genética , Termolisina/química , Termolisina/genética , Valina/fisiología , Acrilatos/metabolismo , Bacillus/enzimología , Caseínas/metabolismo , Dipéptidos/metabolismo , Estabilidad de Enzimas , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hidrólisis , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Estructura Terciaria de Proteína , Termolisina/metabolismoRESUMEN
Most zinc metalloproteinases have the consensus zinc-binding motif sequence HEXXH, in which two histidine residues chelate a catalytic zinc ion. The zinc-binding motif sequence of thermolysin, H(142)ELTH(146), belongs to this motif sequence, while that of dipeptidyl peptidase III (DPP III), H(450)ELLGH(455), belongs to the motif sequence HEXXXH. In this study, we examined effects of conversion of HEXXH to HEXXXH in thermolysin on its activity and stability. Thermolysin variants bearing H(142)ELLGH(146) or H(142)ELTGH(146) (designated T145LG and T145TG respectively) were constructed by site-directed mutagenesis and were produced in Escherichia coli cells by co-expressing the mature and pro domains separately. They did not exhibit hydrolyzing activity for casein or N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but exhibited binding ability to a substrate analog glycyl-D-phenylalanine (Gly-D-Phe). The apparent denaturing temperatures based on the ellipticity at 222 nm of T145LG and T145TG were 85 ± 1 °C and 86 ± 1 °C respectively, almost the same as that of wild-type thermolysin (85 ± 1 °C). These results indicate that conversion of HEXXH to HEXXXH abolishes thermolysin activity, but does not affect its binding ability to Gly-D-Phe or its stability. Our results are in contrast to ones reported previously, that DPP III variants bearing H(450)ELGH(455) exhibit activity.
Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Ingeniería de Proteínas/métodos , Termolisina/química , Termolisina/metabolismo , Zinc/metabolismo , Secuencias de Aminoácidos , Animales , Bovinos , Cobalto/metabolismo , Estabilidad de Enzimas , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Termolisina/genéticaRESUMEN
Neutral salts activate and stabilize thermolysin. We previously found that two single mutations, Asn116âAsp and Asp150âGlu, increase the activity of thermolysin. In the present study, we examined their effects on NaCl-induced activation and stabilization. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, the relative activities (the ratios of the specificity constant, kcat/Km, at x M NaCl to that at 0 M NaCl) at 0.5-4.0 M NaCl of D150E and N116D/D150E were lower than those of wild-type thermolysin (WT) and N116D, respectively. In thermal inactivation at 70 °C, the relative stabilities (the ratios of the first-order rate constant, kobs, at 0 M NaCl to that at x M NaCl) at 0.5-4.0 M NaCl of D150E and N116D/D150E were lower than those of WT and N116D, respectively. These results indicate that unlike Asn116âAsp, Asp150âGlu reduced NaCl-induced activation and stabilization, suggesting that the binding of ions with certain residues of thermolysin is involved in the activation and stabilization.
Asunto(s)
Mutación , Cloruro de Sodio/farmacología , Termolisina/química , Termolisina/metabolismo , Acrilatos/metabolismo , Bacillus/enzimología , Dipéptidos/metabolismo , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Hidrólisis/efectos de los fármacos , Modelos Moleculares , Conformación Proteica , Termolisina/genéticaRESUMEN
The thermolysin variant G8C/N60C/S65P in which the triple mutation in the N-terminal domain, Gly8âCys/Asn60âCys/Ser65âPro, is undertaken increases stability [Yasukawa, K. and Inouye, K. (2007) Improving the activity and stability of thermolysin by site-directed mutagenesis. Biochim. Biophys. Acta 1774, 1281-1288] and its mechanism is examined in this study. The apparent denaturing temperatures based on ellipticity at 222 nm of the wild-type thermolysin (WT), G8C/N60C, S65P and G8C/N60C/S65P were 85, >95, 88 and >95°C, respectively. The first-order rate constants, k(obs), of the thermal inactivation of WT and variants at 10 mM CaCl2 increased with increasing thermal treatment temperatures (70-95°C), and those at 80°C decreased with increasing CaCl2 concentrations (1-100 mM). The k(obs) values were in the order of WT > S65P > G8C/N60CâG8C/N60C/S65P at all temperatures and CaCl2 concentrations. These results indicate that the mutational combination, Gly8âCys/Asn60âCys and Ser65âPro, increases stability only as high as Gly8âCys/Asn60âCys does. Assuming that irreversible inactivation of thermolysin occurs only in the absence of calcium ions, the dissociation constants, K(d), to the calcium ions of WT, G8C/N60C, S65P and G8C/N60C/S65P were 47, 8.9, 17 and 7.2 mM, respectively, suggesting that Gly8âCys/Asn60âCys and Ser65âPro stabilize thermolysin by improving its affinity to calcium ions, most probably the one at the Ca²âº-binding site III in the N-terminal domain.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Mutantes/metabolismo , Termolisina/metabolismo , Sustitución de Aminoácidos , Bacillus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Cloruro de Calcio/química , Dicroismo Circular , Cistina/análisis , Estabilidad de Enzimas , Calor/efectos adversos , Indicadores y Reactivos/química , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Concentración Osmolar , Desnaturalización Proteica , Estructura Terciaria de Proteína , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termolisina/química , Termolisina/genéticaRESUMEN
Ligand functional groups can modulate the contributions of one another to the ligand-protein binding thermodynamics, producing either positive or negative cooperativity. Data presented for four thermolysin phosphonamidate inhibitors demonstrate that the differential binding free energy and enthalpy caused by replacement of a H with a Me group, which binds in the well-hydrated S2' pocket, are more favorable in presence of a ligand carboxylate. The differential entropy is however less favorable. Dissection of these differential thermodynamic parameters, X-ray crystallography, and density-functional theory calculations suggest that these cooperativities are caused by variations in the thermodynamics of the complex hydration shell changes accompanying the HâMe replacement. Specifically, the COO(-) reduces both the enthalpic penalty and the entropic advantage of displacing water molecules from the S2' pocket and causes a subsequent acquisition of a more enthalpically, less entropically, favorable water network. This study contributes to understanding the important role water plays in ligand-protein binding.
Asunto(s)
Termolisina/antagonistas & inhibidores , Termolisina/química , Agua/química , Bacillus/química , Calorimetría , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Modelos Moleculares , Estructura Molecular , Mutación , Organofosfonatos/síntesis química , Organofosfonatos/química , Unión Proteica , Estereoisomerismo , Termodinámica , Termolisina/genéticaRESUMEN
In the N-terminal domain of thermolysin, two anti-parallel ß-strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122 are connected by an Asn116-Gly117 turn to form a ß-hairpin structure. In this study, we examined the role of Asn116 in the activity and stability of thermolysin by site-directed mutagenesis. Of the 19 Asn116 variants, four (N116A, N116D, N116T and N116Q) were produced in Escherichia coli, by co-expressing the mature and pro domains separately, while the other 15 were not. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) at 25°C, the intrinsic k(cat)/K(m) value of N116D was 320% of that of the wild-type thermolysin (WT), and in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM) at pH 7.5 at 25°C, the k(cat)/K(m) value of N116D was 140% of that of WT, indicating that N116D exhibited higher activity than WT. N116Q exhibited similar activity as WT, and N116A and N116T exhibited reduced activities. The first-order rate constants, k(obs), of the thermal inactivation at 80°C were in the order N116A, N116D, N116T > N116Q > WT at all CaCl(2) concentrations examined (1-100 mM), indicating that all variants exhibited reduced stabilities. These results suggest that Asn116 plays an important role in the activity and stability of thermolysin presumably by stabilizing this ß-hairpin structure.
Asunto(s)
Asparagina/genética , Mutagénesis Sitio-Dirigida , Termolisina/química , Termolisina/genética , Secuencia de Aminoácidos , Animales , Bovinos , Estabilidad de Enzimas/genética , Escherichia coli/metabolismo , Fermentación , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/química , Proteínas Mutantes/aislamiento & purificación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Temperatura , Termolisina/metabolismo , Transformación GenéticaRESUMEN
The autodegradation-resistant mutant thermolysins (TLNs), L155A (Leu(155) to Ala) and L155S (Leu(155) to Ser), were previously constructed by site-directed mutagenesis to enhance thermostability. These mutations suppressed autodegradation at position 154-155, resulting in increased thermostability. However, a new autodegradation site became apparent in these mutant TLNs, at position 155-156. In this study, further stabilization of the mutant TLNs to suppress this new autodegradation was attempted by the substitution of Ile(156) to Asp and Val (L155A-I156N, L155A-I156V, L155S-I156N, and L155S-I156V). SDS-PAGE analysis showed that the autodegradation at 155-156 of all double-mutant TLNs was suppressed. Thermostability at 80 °C was enhanced in all double-mutant TLNs (half-life at 80 °C: WT, 18.3 min; L155A, 25.0 min; L155S, 24.0 min; L155A-I156N, 60.8 min; L155A-I156V, 62.4 min; L155S-I156N, 93.3 min; and L155S-I156V, 40.0 min), and k (cat)/K (m) values were: WT, 220; L155A, 240; L155S, 123; L155A-I156N, 62; L155A-I156V, 760; L155S-I156N, 240; and L155S-I156V, 520 min(-1) mM(-1).
Asunto(s)
Bacillus/enzimología , Proteínas Bacterianas/genética , Escherichia coli/genética , Leucina/genética , Termolisina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Bacillus/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Escherichia coli/enzimología , Semivida , Calor , Cinética , Leucina/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termolisina/química , Termolisina/metabolismoRESUMEN
Proteinase-activated receptor 1 (PAR(1)) induces activation of platelet and vascular cells after proteolytic cleavage of its extracellular N terminus by thrombin. In pathological situations, other proteinases may be generated in the circulation and might modify the responses of PAR(1) by cleaving extracellular domains. In this study, epitope-tagged wild-type human PAR(1) (hPAR(1)) and a panel of N-linked glycosylation-deficient mutant receptors were permanently expressed in epithelial cells (Kirsten murine sarcoma virus-transformed rat kidney cells and CHO cells). We have analyzed the role of N-linked glycosylation in regulating proteinase activation/disarming and cell global expression of hPAR(1). We reported for the first time that glycosylation in the N terminus of hPAR(1) downstream of the tethered ligand (especially Asn(75)) governs receptor disarming to trypsin, thermolysin, and the neutrophil proteinases elastase and proteinase 3 but not cathepsin G. In addition, hPAR(1) is heavily N-linked glycosylated and sialylated in epithelial cell lines, and glycosylation occurs at all five consensus sites, namely, Asn(35), Asn(62), Asn(75), Asn(250), and Asn(259). Removing these N-linked glycosylation sequons affected hPAR(1) cell surface expression to varying degrees, and N-linked glycosylation at extracellular loop 2 (especially Asn(250)) of hPAR(1) is essential for optimal receptor cell surface expression and receptor stability.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Mieloblastina/metabolismo , Elastasa Pancreática/metabolismo , Receptor PAR-1/biosíntesis , Animales , Células CHO , Catepsina G/genética , Catepsina G/metabolismo , Línea Celular Transformada , Cricetinae , Cricetulus , Glicosilación , Humanos , Mieloblastina/genética , Elastasa Pancreática/genética , Estructura Terciaria de Proteína , Ratas , Receptor PAR-1/genética , Termolisina/genética , Termolisina/metabolismoRESUMEN
In the N-terminal domain of thermolysin, two polypeptide strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122, are connected by a short loop, Asn116-Gly117, to form an anti-parallel ß-sheet. The Asn112-Trp115 strand is located in the active site, while the Ser118-Tyr122 strand and the Asn116-Gly117 loop are located outside the active site. In this study, we explored the catalytic role of Gly117 by site-directed mutagenesis. Five variants, G117A (Gly117 is replaced by Ala), G117D, G117E, G117K, and G117R, were produced by co-expressing in Escherichia coli the mature and pro domains as independent polypeptides. The production levels were in the order G117E > wild type > G117K, G117R > G117D. G117A was hardly produced. This result is in contrast to our previous one that all 72 active-site thermolysin variants were produced at the similar levels whether they retained activity or not (M. Kusano et al. J. Biochem., 145, 103-113 (2009)). G117E exhibited lower activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and higher activity in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester than the wild-type thermolysin. G117K and G117R exhibited considerably reduced activities. This suggests that Gly117 plays an important role in the activity and stability of thermolysin, presumably by affecting the geometries of the Asn112-Trp115 and Ser118-Tyr122 strands.
Asunto(s)
Biocatálisis , Glicina , Mutagénesis Sitio-Dirigida/métodos , Termolisina/química , Termolisina/metabolismo , Acrilatos/metabolismo , Animales , Dominio Catalítico , Bovinos , Dipéptidos/metabolismo , Estabilidad de Enzimas , Hidrólisis , Modelos Moleculares , Temperatura , Termolisina/genética , Termolisina/aislamiento & purificaciónRESUMEN
We have previously indicated that three single mutations (Leu144-->Ser, Asp150-->Glu, and Ile168-->Ala) in the site-directed mutagenesis of thermolysin increase the activity and two single (Ser53-->Asp and Leu155-->Ala) and one triple (Gly8-->Cys/Asn60-->Cys/Ser65-->Pro) mutations increase the stability. In the present study, aiming to generate highly active and stable thermolysin variants, we combined these mutations and analyzed the effect of combinations on the activity and stability of thermolysin. The combination of the mutations of Leu144-->Ser and Asp150-->Glu yielded the most significant increase in the hydrolytic activities for N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide (FAGLA) and N-carbobenzoxy-L-Asp-L-Phe methyl ester (ZDFM), while that of Leu144-->Ser and Ile168-->Ala abolished the activity. The combination of Ser53-->Asp and Leu155-->Ala yielded the greatest increase in the thermal stability, while that of Ser53-->Asp and Gly8-->Cys/Asn60-->Cys/Ser65-->Pro increased the stability as high as the individual mutations do. The combination of three mutations of Leu144-->Ser, Asp150-->Glu, and Ser53-->Asp yielded a variant L144S/D150E/S53D with improved activity and stability. Its k(cat)/K(m) values in the hydrolysis of FAGLA and ZDFM were 8.6 and 10.2 times higher than those of wild-type thermolysin (WT), respectively, and its rate constant for thermal inactivation at 80 degrees C was 60% of that of WT.
Asunto(s)
Mutagénesis/genética , Mutación/genética , Termolisina/genética , Termolisina/metabolismo , Acrilatos/metabolismo , Caseínas/metabolismo , Dipéptidos/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Escherichia coli , Hidrólisis/efectos de los fármacos , Cinética , Mutagénesis/efectos de los fármacos , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Cloruro de Sodio/farmacología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Especificidad por Sustrato/efectos de los fármacos , Temperatura , Termolisina/química , Termolisina/aislamiento & purificación , Transformación Genética/efectos de los fármacosRESUMEN
The PST-01 protease is highly stable and catalyzes the synthesis of the aspartame precursor with high reaction yields in the presence of organic solvents. However, the synthesis rate using the PST-01 protease was slower than that observed when thermolysin was used. Structural comparison of both enzymes showed particular amino acid differences near the active center. These few residue differences in the PST-01 protease were mutated to match those amino acid types found in thermolysin. The mutated PST-01 proteases at the 114th residue from tyrosine to phenylalanine showed enhancement of synthetic activity. This activity was found to be similar to thermolysin. In addition, mutating the residue in the PST-01 protease with arginine and serine showed more improvement of the activity. The mutant PST-01 protease should be more useful than thermolysin for the synthesis of the aspartame precursor, because this enzyme has higher stability and activity in the presence of organic solvents. The results show the potential of organic solvent-stable enzymes as industrial catalysts.
Asunto(s)
Aspartame/química , Aspartame/metabolismo , Compuestos Orgánicos/farmacología , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Ingeniería de Proteínas , Solventes/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Estabilidad de Enzimas/efectos de los fármacos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Péptido Hidrolasas/química , Conformación Proteica , Especificidad por Sustrato , Termolisina/química , Termolisina/genética , Termolisina/metabolismoRESUMEN
Naturally occurring peptidases from organisms living under extreme conditions are adapted to function in environmental extremes, including temperature, salinity, pH, or pressure. These organisms represent unique sources for new bio-molecules that have both industrial and medicinal application. Adaptive strategies for functioning under extreme conditions are reflected at the enzyme sequence and structural level. Understanding the determinants responsible for unique functional features can be used to enhance the functional features of known proteins. In the present study, the amino acid sequences of 81 peptidases of the thermolysin (M4) family were analyzed for possible determinants of psychrophilic and thermophilic features, by comparing with thermolysin from Bacillus thermoproteolyticus, the prototype enzyme of the family. The analysis indicated that M4 peptidases from cold-adapted species have fewer arginines and more lysines, and also fewer tyrosines and more phenylalanines than the prototype thermolysin. However, the opposite was true for M4 peptidases from thermophilic species. For sequences from thermophilic species the ratio of the seven amino acids I,V,Y,W,R,E,L were correlated to optimal growth temperature.
Asunto(s)
Secuencia de Aminoácidos , Metaloproteasas/química , Metaloproteasas/genética , Adaptación Fisiológica , Aminoácidos/química , Frío , Secuencia de Consenso , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Temperatura , Termolisina/química , Termolisina/genéticaRESUMEN
Protealysin, a protease previously described by us in Serratia proteamaculans, belongs to the group of thermolysin-like proteases (TLPs) that differ from classical TLPs by the precursor structural organization. The propeptide of protealysin precursor has no significant structural similarity to the propeptides of most TLPs. The functions of protealysin-like precursors and mechanisms of their action remain unclear. We studied the pathway of protealysin precursor processing in vitro using standard approaches: modification of the catalytic site and monitoring immobilized precursor maturation. The Glu(113) --> Ala substitution inhibited the precursor maturation, which pointed to the autocatalytic processing. The mutant precursor exposure to active protealysin converted it to the mature enzyme, thus, indicating the intermolecular processing. Intermolecular processing of the mutant protein by other proteases such as thermolysin or subtilisin is also possible. The intact protealysin precursor was efficiently autoprocessed in solution but not after immobilization. These data indicate that the processing of protealysin precursor differs from that of classical TLPs. The protealysin propeptide is cleaved by an autocatalytic or heterocatalytic intermolecular mechanism and is most likely not removed intramolecularly.
Asunto(s)
Proteínas Bacterianas/metabolismo , Precursores de Proteínas/metabolismo , Serratia/enzimología , Termolisina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Vectores Genéticos , Concentración de Iones de Hidrógeno , Cinética , Precursores de Proteínas/genética , Precursores de Proteínas/aislamiento & purificación , Serratia/genética , Termolisina/genética , Termolisina/aislamiento & purificaciónRESUMEN
Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, k(cat)/K(m) (10-40 degrees C), followed the order pseudolysin < MCP-02 < E495 with a ratio of approximately 1:2:4. MCP-02 and E495 have the same optimal temperature (T(opt), 57 degrees C, 5 degrees C lower than pseudolysin) and apparent melting temperature (T(m) = 64 degrees C, approximately 10 degrees C lower than pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.
Asunto(s)
Adaptación Biológica , Biocatálisis , Frío , Cubierta de Hielo/microbiología , Termolisina/química , Termolisina/metabolismo , Zinc/metabolismo , Secuencia de Aminoácidos , Regiones Árticas , Biología Computacional , Simulación por Computador , Secuencia Conservada , Estabilidad de Enzimas , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Océanos y Mares , Pseudomonas/enzimología , Pseudomonas/genética , Alineación de Secuencia , Homología Estructural de Proteína , Termolisina/clasificación , Termolisina/genéticaRESUMEN
Zinc containing peptidases are widely distributed in nature and have important roles in many physiological processes. M4 family comprises numerous zinc-dependent metallopeptidases that hydrolyze peptide bonds. A large number of these enzymes are implicated as virulence factors of the microorganisms that produce them and are therefore potential drug targets. Some enzymes of the family are able to function at the extremes of temperatures, and some function in organic solvents. Thereby enzymes of the thermolysin family have an innovative potential for biotechnological applications.
Asunto(s)
Descubrimiento de Drogas , Termolisina/metabolismo , Termolisina/uso terapéutico , Antibacterianos/metabolismo , Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Humanos , Metaloendopeptidasas/metabolismo , Estructura Molecular , Estructura Terciaria de Proteína , Especificidad por Sustrato , Temperatura , Termolisina/química , Termolisina/genética , Zinc/química , Zinc/metabolismoRESUMEN
The active site of thermolysin is composed of one zinc ion and five polypeptide regions [N-terminal sheet (Asn112-Trp115), alpha-helix 1 (Val139-Thr149), C-terminal loop 1 (Asp150-Gly162), alpha-helix 2 (Ala163-Val176) and C-terminal loop 2 (Gln225-Ser234)]. To explore their catalytic roles, we introduced single amino-acid substitutions into these regions by site-directed mutagenesis and examined their effects on the activity and stability. Seventy variants, in which one of the twelve residues (Ala113, Phe114, Trp115, Asp150, Tyr157, Gly162, Ile168, Ser169, Asp170, Asn227, Val230 and Ser234) was replaced, were produced in Escherichia coli. The hydrolytic activities of thermolysin for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and casein revealed that the N-terminal sheet and alpha-helix 2 were critical in catalysis and the C-terminal loops 1 and 2 were in substrate recognition. Twelve variants were active for both substrates. In the hydrolysis of FAGLA and N-carbobenzoxy-L-Asp-L-Phe methyl ester, the k(cat)/K(m) values of the D150E (in which Asp150 is replaced with Glu) and I168A variants were 2-3 times higher than those of the wild-type (WT) enzyme. Thermal inactivation of thermolysin at 80 degrees C was greatly suppressed with the D150H, D150W, I168A, I168H, N227A, N227H and S234A. The evidence might provide the insights into the activation and stabilization of thermolysin.
Asunto(s)
Péptidos/genética , Termolisina/química , Catálisis , Dominio Catalítico , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Mutagénesis Sitio-Dirigida , Termolisina/genética , Termolisina/metabolismoRESUMEN
The primary structures of the full-length precursors of thermolysin-like proteinases (TLPs) were systemically analyzed. Structural comparison of the precursor amino-terminal regions (ATRs) removed during maturation allowed us to divide the family into two groups: peptidases with short (about 50 amino acids) and long (about 200 amino acids) ATRs. The accumulation of mutations in the ATRs of both types proved to correlate with that in the catalytic domains. No classical signal peptides were identified in the short ATRs, but they contained a conserved PPL-motif near the initiation methionine. The functional role of the short ATRs and PPL-motif is currently unclear. The C-terminal regions (CTRs) of TLP precursors, which are often removed during maturation, too, are found in about a half of precursors with long ATRs, but occur more rarely in precursors with short ATRs. CTRs in TLP precursors contain previously identified conserved domains typical for many other proteins and likely underlie the interaction with high molecular weight substrates.
Asunto(s)
Proteínas Bacterianas/química , Precursores Enzimáticos/química , Péptido Hidrolasas/química , Termolisina/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bases de Datos de Proteínas , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Estructura Terciaria de Proteína , Alineación de Secuencia , Termolisina/genética , Termolisina/metabolismoRESUMEN
Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the k(cat)/K(m) values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl(2) at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl(2) and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca(2+)-binding structures.