tailieunhanh - Báo cáo khoa học: The catalysis of the SARS 3C-like protease is under extensive regulation by its extra domain

The 3C-like protease of the severe acute respiratory syndrome (SARS) cor-onavirus has a C-terminal extra domain in addition to the chymotrypsin-fold adopted by piconavirus 3C proteases hosting the complete catalytic machinery. Previously we identified the extra domain to be involved in enzyme dimerization which has been considered essential for the catalytic activity. | ềFEBS Journal The catalysis of the SARS 3C-like protease is under extensive regulation by its extra domain Jiahai Shi2 and Jianxing Song1 2 1 Department of Biochemistry The Yong Loo Lin Schoolof Medicine National university of Singapore Singapore 2 Department of BiologicalSciences Faculty of Science NationalUniversity of Singapore Singapore Correspondence J. Song Department of BiologicalSciences Faculty of Science NationalUniversity of Singapore 10 Kent Ridge Crescent Singapore 119260 Fax 65 6779 2486 Tel 65 6874 1013 E-mail bchsj@ Received 15 November 2005 revised 22 December 2005 accepted 9 January 2006 doi The 3C-like protease of the severe acute respiratory syndrome SARS coronavirus has a C-terminal extra domain in addition to the chymotrypsin-fold adopted by piconavirus 3C proteases hosting the complete catalytic machinery. Previously we identified the extra domain to be involved in enzyme dimerization which has been considered essential for the catalytic activity. In an initial attempt to map out the extra-domain residues critical for dimerization we have systematically generated 15 point mutations five deletions and one triple mutation and subsequently characterized them by enzymatic assay dynamic light scattering CD and NMR spectroscopy. The results led to identification of four regions critical for enzyme dimerization. Interestingly Asn214Ala mutant with a significant tendency to form a monomer still retained w 30 activity indicating that the relationship between the activity and dimerization might be very complex. Very surprisingly two regions one over Ser284-Thr285-Ile286 and another around Phe291 were discovered on which Ala-mutations significantly increased the enzymatic activities. Based on this a super-active triple-mutant STI A with a activity enhancement was thus engineered by mutating residues Ser284 Thr285 and Ile286 to Ala. The dynamic light scattering CD and NMR characterizations indicate that