3.4.22.70 C184A catalytically inactive 717705 3.4.22.70 C184A mutant enzyme can not cleave the LPXTG motif 647382 3.4.22.70 C184A site-directed mutagenesis 731485 3.4.22.70 C184Hcy site-directed mutagenesis, generation of a sortase mutant with Cys184 replaced by homocysteine (Hcy). Mutant Hcy-sortase is a poor catalyst with less than 1% of wild-type activity. The sensitivity of the active site nucleophiles towards an alkylation reagent correlates with the pKa values of the mutated residues 731485 3.4.22.70 C184S 2700fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 C184Sec site-directed mutagenesis, generation of a sortase mutant with Cys184 replaced by selenocysteine (Sec). Mutant Sec-sortase shows a moderate 2-3fold reduction in catalytic activity. The sensitivity of the active site nucleophiles towards an alkylation reagent correlates with the pKa values of the mutated residues. The pH-profile of mutant Sec-sortase is shifted to more acidic conditions when compared to the wild-type enzyme 731485 3.4.22.70 D170A Tm is 1.7°C higher than the Tm-value of wild-type enzyme. No change in kcat/Km compared to wild-type value 680902 3.4.22.70 D170A turnover-number for o-aminobenzoyl-LPETG-2,4-dinitrophenyl is nearly identical to wild-type value 665718 3.4.22.70 D185A 1.3 fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 D186A 1.8fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 DELTA N59 enhanced solubility of the mutant helps the crystallization 647385 3.4.22.70 DELTA N59 trucation of N-terminal 59 amino acids does not affect the activity of the enzyme 647380, 647385 3.4.22.70 E108A turnover-number for o-aminobenzoyl-LPETG-2,4-dinitrophenyl is 43.8fold lower than wild-type value 665718 3.4.22.70 E171A Tm is 1.8°C lower than the Tm-value of wild-type enzyme. kcat/Km is 5.4fold lower than wild-type value 680902 3.4.22.70 E171A turnover-number for o-aminobenzoyl-LPETG-2,4-dinitrophenyl is 4.7fold lower than wild-type value 665718 3.4.22.70 G167A Tm is 0.5°C lower than the Tm-value of wild-type enzyme. No change in kcat/KM compared to wild-type enzyme 680902 3.4.22.70 H120A 96000fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 H120Q 36000fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 H126A inactive 709123 3.4.22.70 I123G mutant is generated by using wild type truncated protein SrtADELTAN59 as a template. Mutation reduces dimerization 678330 3.4.22.70 I182A mutation produces modest decreases in SrtA activity and leds to substrate inhibition. 28fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 I182S 74fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 K137A mutant is generated by using wild type truncated protein SrtADELTAN59 as a template. Mutation completely disrupts dimerization 678330 3.4.22.70 K152A mutant is generated by using wild type truncated protein SrtADELTAN59 as a template. Mutation increases dimerization 678330 3.4.22.70 K62A mutant is generated by using wild type truncated protein SrtADELTAN59 as a template 678330 3.4.22.70 L169A Tm is 1.8°C lower than the Tm-value of wild-type enzyme. kcat/Km is 93fold lower than wild-type value 680902 3.4.22.70 L181A mutation produces modest decreases in SrtA activity and leds to substrate inhibition. 7.6fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 additional information construction of a mutant strain, DELTASrtA -, 731825 3.4.22.70 additional information construction of the mutant strain TBY-1DELTAsrtA via allelic exchange mutagenesis. The mutant strain has a lower survival capacity in healthy tilapia host blood and it is less virulent than the wild-type strain in tilapia -, 731833 3.4.22.70 additional information marked changes in the specificity profile of SrtA are obtained by replacing the beta6/beta7 loop in SrtA with the corresponding domain from SrtB. The chimeric beta6/beta7 loop swap enzyme (SrtLS) conferrs the ability to acylate NPQTN-containing substrates, with a kcat/Kmapp of 0.0062 /M * s. This enzyme is unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpeptidation to occur. The overall catalytic specificity profile (kcat/Kmapp (NPQTN)/kcat/Kmapp (LPETG)) of SrtLS is altered 700000fold from SrtA. These results indicate that the beta6/beta7 loop is an important site for substrate recognition in sortases 680881 3.4.22.70 additional information srtA mutants AHG263 (srtA gene deleted by allelic replacement with ermC marker) and AHG188 669101 3.4.22.70 additional information srtA-deficient mutant, colonizes the nasopharynx at a significantly lower level than the D39 parent strain during the second and third week of the carriage, and was eliminated from the nasopharynx one week earlier than the D39 pneumococci 668668 3.4.22.70 additional information the enzyme works as a versatile tool in protein engineering. Surface proteins destined for cell wall anchoring contain a LPXTG sequence located in their C-terminus which serves as a substrate recognition motif for the enzyme 731314 3.4.22.70 additional information the F40-sortase mutant loses activity compared to the wild type enzyme and prefers ligation of the FPxTG motif over the native LPxTG sequence. The F40-sortase possesses a remarkable broad selectivity and accepted aromatic amino acids as well as residues with small side chains, including Ala, Asp, Ser, Pro, and Gly, at position 1 of the sorting motif 717705 3.4.22.70 additional information the SrtA variants SrtADELTAN59 and SrtADELTAN25, in which the N-terminal amino acid residues 1-59 and 1-25 of the native enzyme are truncated, perform very differently with regard to the immobilization reaction with green-fluorescent protein (GFPuv) on triglycine-modified polystyrene microbeads by the enzyme. While SrtADELTAN59 efficiently catalyzes the covalent attachment of GFPuv to the surface SrtADELTAN25 is essentially inactive. Besides the length of the N-terminal amino acid extension on the SrtA construct, the position of the His6-tag at either the N- or C-terminus affects the efficiency of enzymatic protein immobilization, overview. Three other mutants of SrtA show rapid initial attachment of the GFPuv target protein to the microbeads, with proceeding reaction time, cleavage of the covalently immobilized target protein from the surface prevails over the coupling reaction 731388 3.4.22.70 N132A mutant is generated by using wild type truncated protein SrtADELTAN59 as a template. Mutation completely disrupts dimerization 678330 3.4.22.70 N98A kcat/Km is 1.2fold lower than wild-type value 665561 3.4.22.70 N98Q kcat/Km is 1.1fold higher than wild-type value 665561 3.4.22.70 P126G mutant is generated by using wild type truncated protein SrtADELTAN59 as a template. Mutation reduces dimerization 678330 3.4.22.70 Q172A Tm is 1.2°C lower than the Tm-value of wild-type enzyme. kcat/Km is 1.4fold lower than wild-type value 680902 3.4.22.70 R197A 540fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 R197A kcat/Km is 2769fold lower than wild-type value 665561 3.4.22.70 R197A Tm is 2.9°C lower than the Tm-value of wild-type enzyme. kcat/Km is 960fold lower than wild-type value 680902 3.4.22.70 R197Cit generation of a semi-synthetic SrtA in which Arg197 is replaced with citrulline, a nonionizable analog. This change results in less than a 3fold decrease in kcat/KM, indicating that Arg197 utilizes a hydrogen bond, rather than an electrostatic interaction. Tm is 0.3°C higher than the Tm-value of wild-type enzyme 680902 3.4.22.70 R197Cit kcat/Km is 5.9fold lower than wild-type value 680902 3.4.22.70 R197H kcat/Km is 610fold lower than wild-type value 665561 3.4.22.70 R197K 1000fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 R197K kcat/Km is 2571fold lower than wild-type value 665561 3.4.22.70 R197K Tm is 2°C lower than the Tm-value of wild-type enzyme. kcat/Km is 690fold lower than wild-type value 680902 3.4.22.70 T180A mutation produces modest decreases in SrtA activity and leds to substrate inhibition. 14fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 T183A 1200fold decrease in kcat/Km compared to wild-type value 678296 3.4.22.70 V168A Tm is 1.7°C lower than the Tm-value of wild-type enzyme. kcat/Km is 5.5fold lower than wild-type value 680902 3.4.22.70 Y143A mutant is generated by using wild type truncated protein SrtADELTAN59 as a template. Mutation completely disrupts dimerization 678330