title: Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128: Comparison to recombinant lysostaphin.
abstract: P128 is a chimeric anti-staphylococcal protein having a catalytic domain from aStaphylococcusbacteriophage K tail associated structural protein and a cell wall targeting domain from theStaphylococcusbacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strainS. simulansbiovarstaphylolyticus, P128 was thermostable and was lytic towardsS. simulansbiovarstaphylolyticusdemonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the Vmax(∆OD600[mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser Vmaxvalue (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent Km(KmAPP) and apparent Kcat(KcatAPP) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin.
authors.affiliation: Molecular Biology Team; Gangagen Biotechnologies Pvt. Ltd.; Bangalore, India.
authors.forename: Sanjeev Rajagopalan
authors.initials: SR
authors.lastname: Saravanan
authors.forename: Vivek Daniel
authors.initials: VD
authors.lastname: Paul
authors.forename: Shilpa
authors.initials: S
authors.lastname: George
authors.forename: Sudarson
authors.initials: S
authors.lastname: Sundarrajan
authors.forename: Nirmal
authors.initials: N
authors.lastname: Kumar
authors.forename: Madhavi
authors.initials: M
authors.lastname: Hebbur
authors.forename: Naveen
authors.initials: N
authors.lastname: Kumar
authors.forename: Ananda
authors.initials: A
authors.lastname: Veena
authors.forename: Uma
authors.initials: U
authors.lastname: Maheshwari
authors.forename: Chemira Biddappa
authors.initials: CB
authors.lastname: Appaiah
authors.forename: Muralidharan
authors.initials: M
authors.lastname: Chidambaran
authors.forename: Anuradha Gopal
authors.initials: AG
authors.lastname: Bhat
authors.forename: Sukumar
authors.initials: S
authors.lastname: Hariharan
authors.forename: Sriram
authors.initials: S
authors.lastname: Padmanabhan
keywords: Staphylococcus aureus; arginine mutation; disulfide bonds; in silico modeling; methicillin resistance; western blot