Research paper LysK, the enzyme lysing Staphylococcus aureus cells: Specific kinetic features and approaches towards stabilization Lyubov Yu. Filatova a , Stephen C. Becker b , David M. Donovan b , Alexander K. Gladilin a , Natalia L. Klyachko a, * a Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Vorobievy Gory,1e11, Moscow 119991, Russia b Animal Biosciences and Biotechnology Laboratory, Animal and Natural Resources Institute, ARS e USDA Beltsville Agricultural Research Center, Beltsville, MD, USA article info Article history: Received 17 September 2009 Accepted 29 January 2010 Available online 6 February 2010 Keywords: Bacteriophage peptidoglycan hydrolase Thermal inactivation of recombinant enzyme LysK endolysin Staphylococci Enzyme stabilization 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 MichaeliseMenten kinetic scheme. Ionization of the enzyme and enzymeesubstrate complex is important for revealing catalytic activity, which 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, pH 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 Ca 2þ cations resulted in an enhanced (more than 100 times) stability of LysK. Dramatic stability decline observed in a narrow temperature range (40e42  C) 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 a crucial role for LysK catalytic activity. Our data on the enzyme activity in the presence of ethylenediaminetetraacetic acid and different metal cations confirmed the importance of metal cation in LysK catalysis. Ó 2010 Elsevier Masson SAS. All rights reserved. 1. Introduction During the last decade, a tendency of increasing the number of cases caused by one of the most dangerous pathogens, staphylo- cocci, has been observed. These infections are among the most widespread and problematic in hospital routine. Thus, in USA more than 100,000 cases of staphylococcal infections are registered per year, which costs annually 4.5e5.7 billion dollars to the country economy. In Ireland in 1999, staphylococci accounted for 31% of the total number of infection-caused diseases, and in 2001 the fraction increased up to 45% [1]. Staphylococcus aureus is especially dangerous: almost 90% of the strains are resistant to penicillin and other antibiotics of penicillinic series [2]. Among the patients infected by methicillin-resistant Staphylococcus aureus (MRSA) strains, the death-rate approaches 31% [3]. All the above-mentioned contribute to an intensive search for new ideology of treatment of patients with staphylococcal infections. Recently it has been found that schemes involving bacteriophage lytic enzymes are among the most perspective [4e6]. Endolysins are bacteriophage encoded enzymes that destroy (lyse) peptidoglycan of cell walls at the terminal stage of phage reproduction cycle in bacterial cells (lysis ”from within”). Endoly- sins are also capable of degrading peptidoglycan when applied externally (as purified recombinant proteins) to the bacterial cell walls, which also results in rapid lysis of bacterial cells (lysis “from without”). The ability of the recombinant enzymes to lyse bacterial cells allows considering them as potential antimicrobial agents [4e8] with a number of important properties, such as extremely high antibacterial activity both in vitro and in vivo, novel mecha- nism of action (enzymatic cleavage of peptydoglycan), activity Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; LysK, the enzyme, lysing staphylococci; OD 600 , optical density at 600 nm; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; CD, circular dichroism; s 1/2 , half-inactivation time; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; mdeg, millidegree. * Corresponding author. Tel.: þ7 495 939 3476; fax: þ7 495 939 5417. E-mail address: Klyachko@enzyme.chem.msu.ru (N.L. Klyachko). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.01.026 Biochimie 92 (2010) 507e513