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ABSTRACT: S1-S4 specificity of the Bacillus licheniformis variant DY proteinase (subtilisin DY) was determined by a series of peptide nitroanilides. The broad S1 specificity is due to the relative flexibility of the binding loop, which exhibits a preference for phenylalanine and accepts poorly the side chains of alanine, valine, lysine, and especially that of glutamic acid, due probably to a steric repulsion by Asn 155 and the narrow entrance of the "pocket." Alanine in position P2 of the substrate is more favorable for the catalysis than glycine. S3 is located on the protein surface. It is more open than the other subsites and can accept a variety of residues. S4 exhibits an extremely high affinity for the aromatic group of phenylalanine. Evidently, hydrophobic forces predominate in the S4--P4 interactions. The results characterize subtilisin DY as a bacterial proteinase with a broad specificity due to the specific geometry and flexibility of the substrate recognition site, which can accommodate different types of amino acid side chains.
Current Microbiology 09/2005; 51(2):71-4. · 1.82 Impact Factor
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ABSTRACT: The inhibition of phospholipase A(2)s (PLA(2)s) is of pharmacological and therapeutic interest because these enzymes are involved in several inflammatory diseases. Elaidoylamide is a powerful inhibitor of a neurotoxic PLA(2) from the Vipera ammodytes meridionalis venom. The X-ray structure of the enzyme-inhibitor complex reveals a new mode of Asp49 PLA(2) inhibition by a fatty acid hydrocarbon chain. The structure contains two identical homodimers in the asymmetric unit. In each dimer one subunit is rotated by 180 degrees with respect to the other and the two molecules are oriented head-to-tail. One molecule of elaidoylamide is bound simultaneously to the substrate binding sites of two associated neurotoxic phospholipase A(2) molecules. The inhibitor binds symmetrically to the hydrophobic channels of the two monomers. The structure can be used to design anti-inflammatory drugs.
Biochemical and Biophysical Research Communications 08/2004; 319(4):1314-21. · 2.48 Impact Factor
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ABSTRACT: Stenotrophomonas maltophilia is increasingly emerging as a multiresistant pathogen in the hospital environment. In immunosuppressed patients, these bacteria may cause severe infections associated with tissue lesions such as pulmonary hemorrhage. This suggests proteolysis as a possible pathogenic mechanism in these infections. This study describes a protease with broad specificity secreted by S. maltophilia. The gene, termed StmPr1, codes for a 63-kDa precursor that is processed to the mature protein of 47 kDa. The enzyme is an alkaline serine protease that, by sequence homology and enzymic properties, can be further classified as a new member of the family of subtilases. It differs from the classic subtilisins in molecular size, in substrate specificity, and probably in the architecture of the active site. The StmPr1 protease is able to degrade several human proteins from serum and connective tissue. Furthermore, pan-protease inhibitors such as alpha(1)-antitrypsin and alpha(2)-macroglobulin were unable to abolish the activity of the bacterial protease. The data support the interpretation that the extracellular protease of S. maltophilia functions as a pathogenic factor and thus could serve as a target for the development of therapeutic agents.
Journal of Biological Chemistry 04/2002; 277(13):11042-9. · 4.77 Impact Factor
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ABSTRACT: Stenotrophomonas maltophilia is increasingly emerging as a multiresistant pathogen in the hospital environment. In immunosuppressed patients, these bacteria
may cause severe infections associated with tissue lesions such as pulmonary hemorrhage. This suggests proteolysis as a possible
pathogenic mechanism in these infections. This study describes a protease with broad specificity secreted by S. maltophilia. The gene, termed StmPr1, codes for a 63-kDa precursor that is processed to the mature protein of 47 kDa. The enzyme is an alkaline serine protease
that, by sequence homology and enzymic properties, can be further classified as a new member of the family of subtilases.
It differs from the classic subtilisins in molecular size, in substrate specificity, and probably in the architecture of the
active site. TheStmPr1 protease is able to degrade several human proteins from serum and connective tissue. Furthermore, pan-protease inhibitors
such as α1-antitrypsin and α2-macroglobulin were unable to abolish the activity of the bacterial protease. The data support the interpretation that the
extracellular protease of S. maltophilia functions as a pathogenic factor and thus could serve as a target for the development of therapeutic agents.
Journal of Biological Chemistry 03/2002; 277(13):11042-11049. · 4.77 Impact Factor
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ABSTRACT: The proteolytic specificity of the neutral Zn-dependent proteinase from Thermoactinomyces sacchari was determined by analysis of the peptides obtained after incubation with the oxidized insulin B chain as a substrate. The
enzyme is an endopeptidase with broad specificity. In total, 12 peptide bonds in the B chain of insulin were hydrolyzed. The
major requirement is that a hydrophobic residue such as Leu, Val, or Phe should participate with the α-amino group in the
bond to be cleaved. However, hydrolysis of bonds at the N-terminal side of His, Thr, and Gly was also observed. The peptide
bond Leu 15–Tyr 16 in the oxidized insulin B chain, which is the major cleavage site for the alkaline microbial proteinases,
is resistant to the attacks of the enzyme from Thermoactinomyces sacchari and other neutral proteinases. The proteolytic activity of the Zn-dependent proteinase from T. sacchari is different from those of other metalloendopeptidases from microorganisms.
Current Microbiology 06/2000; 41(1):70-72. · 1.82 Impact Factor
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ABSTRACT: Disulfide bonds and calcium ions contribute significantly to the stability of the hemocyanin from the mollusc Rapana thomasiana grosse (gastropod). An extremely powerful protective effect of Ca2+ at a concentration of 100 mM (100% protection) against the destructive effect of reductants like dithiothreitol was observed. This is important for the practical application of molluscan hemocyanins in experimental biochemistry, immunology and medicine. The reduction of the disulfide bonds in the Rapana hemocyanin leads to a 20% decrease of the a-helical structure. The S-S bonds contribute significantly to the free energy of stabilization in water increasing delta G(D)H2O by 6.9 kJ mol (-1) The data are related to the X-ray model of the Rapana hemocyanin functional unit RtH2e. The results of this study can be of common validity for related respiratory proteins because the cysteine residues are conserved in all sequences of molluscan hemocyanins published so far.
Zeitschrift fur Naturforschung C 59(3-4):281-7. · 0.77 Impact Factor
