Probing the Role of Divalent Metal Ions in a Bacterial Psychrophilic Metalloprotease: Binding Studies of an Enzyme in the Crystalline State by X-Ray Crystallography (original) (raw)
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Proteins-structure Function and Bioinformatics, 2003
Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. It is in general accepted that psychrophilic enzymes are more flexible to allow easy accommodation and transformation of the substrates at low energy costs. Here, we report the structures of two crystal forms of the alkaline protease from an Antarctic Pseudomonas species (PAP), solved to 2.1-and 1.96-Å resolution, respectively. Comparative studies of PAP structures with mesophilic counterparts show that the overall structures are similar but that the conformation of the substrate-free active site in PAP resembles that of the substrate-bound region of the mesophilic homolog, with both an active-site tyrosine and a substrate-binding loop displaying a conformation as in the substrate-bound form of the mesophilic proteases. Further, a region in the catalytic domain of PAP undergoes a conformational change with a loop movement as large as 13 Å, induced by the binding of an extra calcium ion. Finally, the active site is more accessible due to deletions occurring in surrounding loop regions. Proteins 2003;50:636 -647.
Biochemical and biophysical research communications, 2014
The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co(2+) rather than Zn(2+): the kcat (s(-1)) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co(2+), and Zn(2+)conditions, respectively. Consistently, addition of low concentrations of Co(2+) to PaAP previously saturated with Zn(2+) greatly enhanced the enzymatic activity, suggesting that Co(2+)may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co(2+) or Zn(2+) commonly showed two metal ions in the active site coordinated with three conserved residues and a bic...
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
Journal of Biotechnology, 1992
A psychrophilic Pseudomonas fluorescens forming protease was isolated from glacier materials. The largest amount of protease was excreted at a cultivation temperature of 10°C after 96 h. The purified enzyme was a metalloprotease (inhibition by EDTA) and had a molecular mass of approximately 50 kDa and an isoelectric point of 7.4-7.5. Maximal activity towards azocasein was observed at 40°C and pH 6.0-7.0. The enzyme showed a high thermo-instability and a low activation energy. Only up to a casein concentration of 0.4% a Michaelis-Menten kinetics was observed.
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.
In silico characterization and structural modeling of bacterial metalloprotease of family M4
Journal of Genetic Engineering & Biotechnology, 2021
Background The M4 family of metalloproteases is comprised of a large number of zinc-containing metalloproteases. A large number of these enzymes are important virulence factors of pathogenic bacteria and therefore potential drug targets. Whereas some enzymes have potential for biotechnological applications, the M4 family of metalloproteases is known almost exclusively from bacteria. The aim of the study was to identify the structure and properties of M4 metalloprotease proteins. Results A total of 31 protein sequences of M4 metalloprotease retrieved from UniProt representing different species of bacteria have been characterized for various physiochemical properties. They were thermostable, hydrophillic protein of a molecular mass ranging from 38 to 66 KDa. Correlation on the basis of both enzymes and respective genes has also been studied by phylogenetic tree. B. cereus M4 metalloprotease (PDB ID: 1NPC) was selected as a representative species for secondary and tertiary structures a...
Iranian Journal of Biotechnology, 2012
A novel neutrophilic metalloprotease was isolated from Pseudomonas sp. DR89 isolate which was identified in a mineral spring in Iran. The enzyme was purified from the isolate to 21-folds in a three-step procedure involving ammonium sulfate precipitation, Q-Sepharose ionic exchange and Sephadex G-100 gel filtration chromatography. Resuts showed that the enzyme was active at high temperatures and in a wide-range pH of 5-11 with the optimum of 8.0. The zymogram and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed the presence of one protease with a molecular weight of 74 kDa. The enzyme activity was decreased by Zn2+, Mn2+, H 2 O 2 and cetyl trimethylammonium bromide (CTAB), whereas its activity was increased by Ca2+, Mg2+, Cu2+ and dimethyl sulfoxide (DMSO). Na + , phenylmethyl sulfonylfluoride (PMSF), β-mercaptoethanol, sodium dodecyl sulfate (SDS), and Triton X-100 did not show a considerable effect on its activity. Casein was a better substrate than bovin...
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.
International Journal of Molecular Sciences, 2012
Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 °C and