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United States Department of Agriculture

Agricultural Research Service

Research Project: EVALUATION OF GENETICALLY ENGINEERED CATTLE AND REFINING TECHNIQUES FOR PRODUCING THEM

Location: Animal Biosciences and Biotechnology Laboratory

Title: LysK, the enzyme lysing Staphylococcus aureus cells: specific kinetic features and approaches towards stabilization

Authors
item Filatova, Lyubov -
item DONOVAN, DAVID
item BECKER, STEPHEN
item Klyachko, Natalia -
item Gladilin, Alexander -

Submitted to: Biochimie
Publication Type: Review Article
Publication Acceptance Date: January 29, 2010
Publication Date: February 6, 2010
Citation: Filatova, L.Y., Becker, S.C., Donovan, D.M., Gladilin, A.K., Klyachko, N.L. 2010. LysK, the enzyme lysing Staphylococcus aureus cells: specific kinetic features and approaches towards stabilization. Biochimie. 92(5):507-13.

Interpretive Summary: C. Problem -- There is a need for novel antimicrobials that might lyse multi-drug resistant and biofilm forms of staphylococcal pathogens. Ideally, new antimicrobials will not be broad range, but rather specific to the targeted pathogen, thus avoiding the likelihood of resistance development in exposed commensal bacteria. C. Accomplishment -- LysK is a peptidoglycan hydrolase with high specificity to all forms of staphylococcal pathogens included biofilms. Thus, the enzyme might prove to be a much needed novel protein/enzyme antimicrobial that is can be considered a prospective new class of antimicrobial agent. Knowledge of LysK properties and behavior would allow optimizing conditions of its storage as well as formulating strategy towards its stabilization. C. Contribution of Accomplishment to Solving the Problem – Our data on the enzyme activity, kinetics, importance of metal cations and stability are important contributions to understanding the limitations of the enzyme and optimal conditions for its potential use as an antimicrobial.

Technical Abstract: LysK, the enzyme lysing cells of Staphylococcus aureus, can be considered as perspective antimicrobial agent. Knowledge of LysK properties and behavior would allow optimizing conditions of its storage as well as formulating strategy towards its stabilization. Reaction of LysK with substrate (suspension of autoclaved Staphylococcus aureus cells) has been found to be adequately described by the two-stage Michaelis-Menten kinetic scheme. Ionization of the enzyme and enzyme-substrate complex important for revealing catalytic activity is controlled by two ionogenic groups with pK 6.0 and 9.6. Ionization energy of the group with pK 6.0 is of 30 kJ/mol, thus, pointing out on His residue; pK 9.6 might be attributed to metal ion or metal-bound water molecule. At temperatures lower than 40°C, LysK stability depends on its concentration, '' and presence of low molecular weight additives. Results of electrophoresis under native and denaturing conditions as well as sedimentation analysis strongly suggest that aggregation is behind LysK inactivation. Decrease in the enzyme concentration, as well as addition of low molecular mass polyols (glycerol, sorbitol, sucrose, trehalose) and Ca2+ cations resulted in an enhanced (more than 100 times) stability of LysK. Dramatic stability decline observed in a narrow temperature range (40 - 42oC) was accompanied by changes in LysK secondary structure as confirmed by CD spectroscopy studies. According to computer modeling data, Cys and His residues and metal cation might play crucial role for LysK catalytic activity.

Last Modified: 9/29/2014
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