Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases (original) (raw)
Related papers
Journal of Bacteriology, 2003
The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated in Antarctica belongs to the clan of metzincins, for which a zinc ion is essential for catalytic activity. Binding studies in the crystalline state have been performed by X-ray crystallography in order to improve the understanding of the role of the zinc and calcium ions bound to this protease. Cocrystallization and soaking experiments with EDTA in a concentration range from 1 to 85 mM have resulted in five three-dimensional structures with a distinct number of metal ions occupying the ion-binding sites. Evolution of the structural changes observed in the vicinity of each cation-binding site has been studied as a function of the concentration of EDTA, as well as of time, in the presence of the chelator. Among others, we have found that the catalytic zinc ion was the first ion to be chelated, ahead of a weakly bound calcium ion (Ca 700) exclusive to the psychrophilic enzyme. Upon removal of the catalytic zinc ion, the side chains of the active-site residues His-173, His-179 and Tyr-209 shifted ϳ4, 1.0, and 1.6 Å, respectively. Our studies confirm and also explain the sensitivity of PAP toward moderate EDTA concentrations and propose distinct roles for the calcium ions. A new crystal form of native PAP validates our previous predictions regarding the adaptation of this enzyme to cold environments as well as the proteolytic domain calcium ion being exclusive for PAP independent of crystallization conditions.
AIMS Bioengineering, 2017
Cold-adapted enzymes are generally derived from psychrophilic microorganisms and have features that make them very attractive for industrial and biotechnological purposes. In this work, we identified a 50 kDa extracellular protease (MP10) from the Antarctic isolate Pseudomonas sp. AU10. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and partially characterized. MP10 is an alkaline thermosensitive serinemetallo protease with optimal activity at pH 8.0 and 40 ℃, in the presence of 1.5 mM Ca 2+. MP10 showed 100% residual activity and stability (up to 60 min) when incubated with 7% of non-ionic surfactants (Triton X-100, Tween-80 and Tween-20) and 1.5% of the oxidizing agent hydrogen peroxide. The 3D MP10 structure was predicted and compared with the crystal structure of mesophilic homologous protease produced by Pseudomonas aeruginosa PA01 (reference strain) and other proteases, showing similarity in surface area and volume of proteins, but a significantly higher surface pocket area and volume of MP10. The observed differences presumably may explain the enhanced activity of MP10 for substrate binding at low temperatures. These results give insight to the potential use of MP10 in developing new biotechnologically processes active at low to moderate temperatures, probably with focus in the detergent industry.
AIMS Bioengineering, 2017
Cold-adapted enzymes are generally derived from psychrophilic microorganisms and have features that make them very attractive for industrial and biotechnological purposes. In this work, we identified a 50 kDa extracellular protease (MP10) from the Antarctic isolate Pseudomonas sp. AU10. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and partially characterized. MP10 is an alkaline thermosensitive serinemetallo protease with optimal activity at pH 8.0 and 40 ℃, in the presence of 1.5 mM Ca 2+. MP10 showed 100% residual activity and stability (up to 60 min) when incubated with 7% of non-ionic surfactants (Triton X-100, Tween-80 and Tween-20) and 1.5% of the oxidizing agent hydrogen peroxide. The 3D MP10 structure was predicted and compared with the crystal structure of mesophilic homologous protease produced by Pseudomonas aeruginosa PA01 (reference strain) and other proteases, showing similarity in surface area and volume of proteins, but a significantly higher surface pocket area and volume of MP10. The observed differences presumably may explain the enhanced activity of MP10 for substrate binding at low temperatures. These results give insight to the potential use of MP10 in developing new biotechnologically processes active at low to moderate temperatures, probably with focus in the detergent industry.
Crystal structure of an alkaline protease from Bacillus alcalophilus at 2.4Åresolution
FEBS Letters, 1990
The crystal structure of an alkaline protease from Bacillus alcalophilus has been determined by X-ray diffraction at 2.4 ,~ resolution. The enzyme crystallizes in space group P212~21 with lattice constants a= 53.7, b= 61.6, c=75.9 .&. The structure was solved by molecular replacement using the structure of subtilisin Carlsberg as search model. Refinement using molecular dynamics and restrained least squares methods results in a crystallographic R-factor of 0.185. The tertiary structure is very similar to that of subtilisin Carlsberg. The greatest structural differences occur in loops at the surface of the protein.
tMetalloproteases represent the largest fraction of the global enzyme market. For biotechnological pur-poses the accumulation of product (i.e. productivity) provides the best measure of assessing enzymeperformance because it takes into account the interplay between activity, stability, activation and inhi-bition. Studies assessing the productivity of alkaline metalloproteases and chemicals that improve theirproductivity have not previously been reported. In this study we report the specificity, productiv-ity, kinetic and thermodynamic properties of an extracellular protease, purified from a new strain ofPseudomonas sp. isolated from refrigerated milk. Mass spectrometry analysis revealed the enzyme is aserralysin-type alkaline metalloprotease, with broad cleavage-site specificity. By studying the effects ofCa2+ion removal (using a chelator) and Ca2+ion addition, conditions were identified that led to an increasein productivity by 300% (6.3 vs 1.9 mg azopeptide g−1enzyme at 40◦C). The basis for the enhanced pro-ductivity was linked to elevated melting temperatures of secondary (Tm47 vs 38◦C) and tertiary structure(Tm50 vs 44◦C), increased half-life of inactivation (t1/230 vs 4.9 min), increased optimum temperature(44 vs 36◦C), and changes in both catalytic activity (kcat3.3 vs 2.2 min−1) and substrate affinity (Km3.9vs 2.5 mg mL−1). Thermodynamic data were indicative of Ca2+-binding causing the transition-state to bemore ordered (less entropy) relative to the folded-state, thereby resisting a transition to an unfoldedstate. The specificity, kinetics and response to calcium of this AMP illustrate its potential usefulness forindustrial applications, and the research highlights the broader potential for using calcium to enhancethe productivity of proteases.
Psychrophilic enzymes: a thermodynamic challenge
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1997
Psychrophilic microorganisms, hosts of permanently cold habitats, produce enzymes which are adapted to work at low temperatures. When compared to their mesophilic counterparts, these enzymes display a higher catalytic efficiency over a temperature range of roughly 0-308C and a high thermosensitivity. The molecular characteristics of cold enzymes originating from Antarctic bacteria have been approached through protein modelling and X-ray crystallography. The deduced three-dimensional structures of cold a-amylase, b-lactamase, lipase and subtilisin have been compared to their mesophilic homologs. It appears that the molecular adaptation resides in a weakening of the intramolecular interactions, and in some cases in an increase of the interaction with the solvent, leading to more flexible molecular edifices capable of performing catalysis at a lower energy cost. q 1997 Elsevier Science B.V.
JBIC Journal of Biological Inorganic Chemistry, 2006
The aminopeptidase from Aeromonas proteolytica (AAP) contains two zinc ions in the active site and catalyzes the degradation of peptides. Herein we report the crystal structures of AAP at 0.95-Å resolution at neutral pH and at 1.24-Å resolution at low pH. The combination of these structures allowed the precise modeling of atomic positions, the identification of the metal bridging oxygen species, and insight into the physical properties of the metal ions. On the basis of these structures, a new putative catalytic mechanism is proposed for AAP that is likely relevant to all binuclear metalloproteases.
Biotechnology Progress, 2012
The purification and characterization of psychro-thermoalkalistable protease from psychrotrophic Pseudomonas putida isolate is being reported for the first time. A $53 kDa protease was purified 21.4-folds with 57.2% recovery by ultrafiltration and hydrophobic interaction chromatography. Kinetic analyses revealed the K m and V max to be 1.169 mg mL À1 and 0.833 mg mL À1 min À1 , respectively. The k cat value of 3.05 Â 10 2 s À1 indicated high affinity and catalytic efficiency toward casein. The protease was most active at pH 9.5 and 40 C, with 100% stability in pH and temperature range of 6.0-11.0 and 10-40 C, respectively. Presence of Zn 2þ increased the thermostability of protease (at 70 C) by 433%. Ethylene diamine tetra acetic acid (EDTA) and 1,10-phenanthroline were inhibitory, whereas phenyl methyl sulfonyl fluoride (PMSF), p-chloro mercuric benzoate (PCMB), and b-mercaptoethanol were ineffective, revealing the enzyme to be a metalloprotease. Zinc, calcium, iron, nickel, and copper at 1 mM increased the enzyme activity (102-134%). Complete reversion of enzyme inhibition (caused by Ethylene diamine tetra acetic acid [EDTA]) by Zn 2þ affirmed this enzyme as zinc-dependent metalloprotease. At 0.1% concentration, Triton X-100 and Tween 80 slightly increased, while SDS and H 2 O 2 reduced the protease activity. In the presence of 0.1% commercial detergents, the enzyme was fairly stable (54-81%). In the presence of organic solvent, the protease was remarkably stable exhibiting 72-191% activities. In contrast, savinase exhibited good stability in the presence of hydrophilic solvents, while chymotrypsin showed elevated activities with benzene, toluene, and xylene only. Circular dichroism analysis revealed the protease as a b-rich protein, having large fraction
Structural and biochemical characterization of a novel thermophilic Coh01147 protease
PLOS ONE, 2020
Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, K m , and k cat , k ca t/K m values of 13.72 mM, 3.143 × 10 −3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60˚C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t 1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100˚C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.
Protein Engineering Design & Selection, 2010
Edited by Taiji Imoto Stabilizing an enzyme while improving its activity may be difficult with respect to a general trade off relation between stability and function at the level of individual mutations. We have used site-directed mutagenesis to investigate the possibility of parallel improvements of thermostability and activity in the neutral protease from Salinovibrio proteolyticus. Four out of seven point mutations are able to promote both activity and thermostability individually and combinedly. The catalytic efficiency (k cat /K m) of four-amino acid substituted variant (quadruple mutant) at 608 8 8 8 8C is 18-fold higher than wild type, whereas at optimum temperature is almost 50-fold higher. Quadruple mutant shows an upward shift of 148 8 8 8 8C in the temperature optimum, and a 20-, 24-, 7-and 5-fold increase in half-life at 60, 65, 70 and 758 8 8 8 8C, respectively, as a result of enhanced calcium binding. Theoretical studies have provided evidences that hinge-bending angle is reduced by amino acid substitutions. Finally, we conclude that the extended surface region between residues 187-228, which involves three out of four beneficial mutations, influences the hinge angle which is determinant for catalysis and also involves the structural calcium which is critical for stability.