title: Sequence and kinetic analyses of streptokinase from two group G streptococci with high fibrin-dependent plasminogen activities and the identification of novel altered amino acids as potential hot spots.
abstract: OBJECTIVE
  To gain insights on the degree of heterogeneity and kinetic differences of streptokinase (SK) from group G (SKG) Streptococci compared with standard SK from group C (SKC) and identification of potentially contributing critical residues (hotspots).
  
  
  RESULTS
  DNA and sequencing analyses confirmed the proper construction of all SK encoding vectors (two SKGs and one standard SKC). SDS-PAGE and western blot analyses confirmed the expression and proper purification of the recombinant SKs from E.coli with the expected size of 47 kDa. Kinetic analyses of two SKGs, compared with SKC, showed higher levels of specific [(×103IU/mg) of 725 and 715 vs. 536] and fibrin-dependent proteolytic activities [Kcat/KM(min-1/µM) of 37 and 30 vs. 23], accompanied by declined fibrin-independent amidolytic activities [Kcat/KM(min-1/mM) of 109 and 84 vs. 113], respectively. Sequence alignments identified 10 novel residual substitutions scattered in SKα (I33F, R45Q, SKG132, A47D, and G55 N), SKβ (N228 K, F287I), and SKγ domains (L335 V, V396A, T403S) of SKGs, as potential hotspots.
  
  
  CONCLUSIONS
  The residue substitutions identified might critically contribute as hot spots to different kinetic parameters of SKGs and might assist in further elucidation of structure/function relations and rational design of SKs with improved (fibrin-dependent) therapeutic properties.
authors.affiliation: Pilot of Nano-biotechnology Department, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Malihe
authors.initials: M
authors.lastname: Keramati
authors.affiliation: Microbiology Department, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mohammad Mehdi
authors.initials: MM
authors.lastname: Aslani
authors.affiliation: Biochemistry Department, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Shohreh
authors.initials: S
authors.lastname: Khatami
authors.affiliation: Virology Department, Pasteur Institute of Iran, No. 69, Pasteur Ave, Tehran, P.O. Box: 1316943551, Iran. farzin.roohvand2@gmail.com.
authors.forename: Farzin
authors.identifier: http://orcid.org/0000-0002-9597-3555
authors.initials: F
authors.lastname: Roohvand
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D005337:Fibrin / Q000378:metabolism; D007700:Kinetics; D010958:Plasminogen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics; D013300:Streptokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005337:Fibrin; D010958:Plasminogen; D013300:Streptokinase
keywords: Amidolytic activity; Group G streptococci; Hot spots; Kinetic assay; Proteolytic activity; SK variant; Streptokinase