Purification, crystallization and preliminary X-ray investigation of aqualysin I, a heat-stable serine protease

Green, P. R.; Oliver, J D; Strickland, L. C.; Toerner, D. R.; Matsuzawa, Hidenori; Ohta, Takao

doi:10.1107/s0907444992012083

Cited by 9 publications

(6 citation statements)

References 5 publications

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“…Table 1 lists the salt bridge-forming amino acid residues in AQN, VPR and Serratia proteinase K-like enzyme (SPRK; (Larsen et al [ 2006 ])), which were identified based on the known structures of AQN (4DZT; (Green et al [ 1993 ])), VPR (1SH7; (Arnórsdóttir et al [ 2005 ])) and SPRK (2B6N; (Helland et al [ 2006 ])), respectively. There are three conserved salt bridges in both AQN and VPR (Figure 1 a and b).…”

Section: Resultsmentioning

confidence: 99%

“…In VPR, a psychrophilic enzyme, seven salt bridges (Arg10-Asp183, Arg14-Asp274, Asp56-Arg95, Asp59-Arg95, Asp138-Arg169, Arg185-Asp260 and Glu236-Arg252) have been identified in the known structure of the enzyme (PDB accession number: 1SH7; (Arnórsdóttir et al [ 2005 ])). However, AQN has six salt bridges (Arg12-Asp183, Asp17-Arg259, Arg31-Asp237, Arg43-Asp212, Asp58-Arg95 and Asp138-Arg169) in its structure (4DZT; (Green et al [ 1993 ])) (Table 1 ). Both enzymes have a similar number of salt bridges; however, their thermal stabilities are quite different.…”

Section: Introductionmentioning

confidence: 99%

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Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1

et al. 2014

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The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1

et al. 2014

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“…The 3D structure of AsP has been predicted using DS‐MODELLER (http://salilab.org/modeller) and PHYRE2 (http://www.sbg.bio.ic.ac.uk/). From the homology modeling searching, two templates were selected: high‐resolution X‐ray crystallography structure of the T. aquaticus 4DZT and 3F70 . Out of the two models, 4DZT was found to be the best model according to the scoring of PROCHECK, totally non‐local energy of the protein ( E/kT units) and overall model quality Z‐score.…”

Section: Methodsmentioning

confidence: 99%

Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus,Alternaria solani

Chandrasekaran

Chandrasekar

et al. 2013

J. Basic Microbiol.

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Serine proteases are involved in an enormous number of biological processes. The present study aims at characterizing three‐dimensional (3D) molecular architecture of serine proteases from early blight pathogen, Alternaria solani that are hypothesized to be markers of phytopathogenicity. A serine protease was purified to homogeneity and MALDI‐TOF‐MS/MS analysis revealed that protease produced by A. solani belongs to alkaline serine proteases (AsP). AsP is made up of 403 amino acid residues with molecular weight of 42.1 kDa (Isoelectric point – 6.51) and its molecular formula was C1859H2930N516O595S4. AsP structure model was built based on its comparative homology with serine protease using the program, MODELER. AsP had 16 β‐sheets and 10 α‐helices, with Ser350 (G347–G357), Asp158 (D158–H169), and His193 (H193–G203) in separate turn/coil structures. Biological metal binding region situated near 6th‐helix and His193 residue is responsible for metal binding site. Also, calcium ion (Ca2+) is coordinated by the carboxyl groups of Lys84, Ile85, Lys86, Asp87, Phe88, Ala89, Ala90 (K84–A90) for first Ca2+ binding site and carbonyl oxygen atom of Lys244, Gly245, Arg246, Thr247, Lys248, Lys249, and Ala250 (K244–A250), for second Ca2+ binding site. Moreover, Ramachandran plot analysis of protein residues falling into most favored secondary structures were determined (83.3%). The predicted molecular 3D structural model was further verified using PROCHECK, ERRAT, and VADAR servers to confirm the geometry and stereo‐chemical parameters of the molecular structural design. The functional analysis of AsP 3D molecular structure predictions familiar in the current study may provide a new perspective in the understanding and identification of antifungal protease inhibitor designing.

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“…Also, the thermostable enzymes should be at least as likely to give well diffracting crystals as the Vibrio-proteinase. In fact, preliminary X-ray crystallographic analysis on aqualysin I was published in 1993 (Green et al, 1993), but was never finished. Green et al, (1993) reported that no molecular replacement solution was found using proteinase K as a search model.…”

Section: Discussionmentioning

confidence: 99%

“…In fact, preliminary X-ray crystallographic analysis on aqualysin I was published in 1993 (Green et al, 1993), but was never finished. Green et al, (1993) reported that no molecular replacement solution was found using proteinase K as a search model. This would probably not be a problem now that the much more similar crystal structure of the Vibrio-proteinase is available.…”

Section: Discussionmentioning

confidence: 99%

Crystallographic studies on a cold adapted subtilase and proteins involved in mRNA processing

Arnórsdóttir¹

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The crystal structure of a subtilisin-like serine proteinase from the psychrotrophic marine bacterium, Vibrio sp. PA-44, was solved by means of molecular replacement and refined at 1.84 Å resolution. This is the first structure of a cold adapted subtilase to be determined and its elucidation facilitates examination of the molecular principles underlying temperature adaptation of enzymes. The cold adapted Vibrio-proteinase was compared to known three-dimensional structures of homologous enzymes of meso-and thermophilic origin, proteinase K and thermitase, to which it has high structural resemblance. The main structural features standing out as plausible determinants of the different temperature adaptation of the enzymes involve the character of their exposed and buried surface areas. The hydrophobic effect is found to play a significant role for the structural stability of the meso-and thermophile enzymes, whereas the cold enzyme exposes more of its apolar surface. In addition, the cold adapted Vibrio-proteinase is distinguished from the more stable enzymes by its strong anionic character arising from the high occurrence of uncompensated negatively charged residues on its surface.Interestingly, both the cold and thermophile proteinases differ from the mesophile enzyme in having more extensive hydrogen-and ion pair interactions in their structures, supporting suggestions of the dual role of electrostatic interactions in adaptation of enzymes to both high and low temperatures. The Vibrio-proteinase has three calcium ions associated with its structure, one of which is in a calcium-binding site not described in other subtilases.A 61 kDa protein component of the human spliceosome is indispensable for the assembly of the [U4/U6•U5] triple snRNP. The gene encoding the 61 kDa spliceosomal protein was overexpressed in E. coli and purified for crystallisation trials. No crystals were obtained. Evidence of incomplete folding and heterogeneity due to unspecific binding of E. coli nucleic acids emerged in the purification process.A 42 kDa protein component of the Trypanosoma brucei RNA editosome has endo-and exonuclease activity and is suggested to mediate protein-protein and/or protein-RNA interactions. Attempts were made to crystallise the 42 kDa protein with and without non-hydrolysable double stranded nucleic acid ligands but without success.Protein preparation for crystallisation trials of p61 .

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Purification, crystallization and preliminary X-ray investigation of aqualysin I, a heat-stable serine protease

Abstract: Aqualysin I, a thermostable protease found in the culture medium of Thermus aquaticus YT-1, has been purified to homogeneity using a combination of ion-exchange and affinity chromatography. It is a polypeptide with a molecular weight of 28 350 . Eur. J. Biochem. 173, 491497]

Cited by 9 publications

References 5 publications

Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1

Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1

Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus,Alternaria solani

Crystallographic studies on a cold adapted subtilase and proteins involved in mRNA processing

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