Streptococcus pneumoniae hyaluronate lyase contains two non-cooperative independent folding/unfolding structural domains: characterization of functional domain and inhibitors of enzyme.
Hyaluronate lyase contributes directly to bacterial invasion by degrading hyaluronan, the major component of host extracellular matrix of connective tissues. Streptococcus pneumoniae hyaluronate lyase (SpnHL) is built from two structural domains that interact through interface residues, in addition to being connected by a peptide linker. For the first time we demonstrate that the N- and C-terminal domains of SpnHL fold/unfold independent of each other suggesting the absence of any significant cooperative interactions between them. The C-terminal domain of SpnHL is less stable than the N-terminal domain against thermal and guanidine hydrochloride denaturation. The intact N-terminal domain was purified after limited proteolysis of SpnHL under conditions where only the C-terminal domain was unfolded. Isolated N-terminal domain of SpnHL had similar thermal stability as when present in the native enzyme and was found to be enzymatically active demonstrating that it is capable of carrying out enzymatic reaction on its own. Functional studies demonstrated that guanidine hydrochloride, guanidine isothiocyanate, l-arginine methyl ester, and l-arginine inhibit the enzymatic activity of SpnHL at very low concentrations. This provides a lead for new chemical entities that can be exploited for designing effective inhibitors of SpnHL.
Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India.
Md Sohail
MS
Akhtar
Vinod
V
Bhakuni
D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D017879:Isothiocyanates / Q000494:pharmacology; D015636:Magnesium Chloride / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D011133:Polysaccharide-Lyases / Q000737:chemistry*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013696:Temperature
D006146:Guanidines; D017879:Isothiocyanates; C054435:guanidine isothiocyanate; D015636:Magnesium Chloride; C026512:arginine methyl ester; D001120:Arginine; D011133:Polysaccharide-Lyases; C100002:hyaluronate lyase; D019791:Guanidine; D002122:Calcium Chloride; D019331:NG-Nitroarginine Methyl Ester