The bacterial degradation of polychlorinated biphenyls depends on the ability of the enzyme biphenyl 2,3-dioxygenase (BPDO) to catalyze their oxygenation. Analysis of hybrid BPDOs obtained using common restriction sites to exchange large DNA fragments between LB400 bphA and B-356 bphA showed that the C-terminal portion of LB400 alpha subunit can withstand extensive structural modifications, and that these modifications can change the catalytic properties of the enzyme. On the other hand, exchanging the C-terminal portion of B-356 BPDO alpha subunit with that of LB400 alpha subunit generated inactive chimeras. Data encourage an enzyme engineering approach, consisting of introducing extensive modifications of the C-terminal portion of LB400 bphA to extend BPDO catalytic properties toward polychlorinated biphenyls.

The 49-kDa, nuclear inclusion a-like, viral protein genome-linked proteinase (VPg-Pro) of turnip mosaic potyvirus (TuMV) was expressed in Escherichia coli. The protein was produced in a soluble form at high levels and was active, as demonstrated by intermolecular cleavage of the polymerase capsid protein (Pol-CP) substrate. The VPg-Pro was purified by metal-chelation and ion-exchange chromatographies. Two forms of VPg-Pro, which differed in molecular masses, were obtained during isolation; their identities were confirmed by immunoblot analysis and N-terminal amino acid sequencing. Data indicated that cleavage took place at a site near the C-terminus of VPg-Pro and was the result of the proteolytic activity of the viral protein. The purified proteinase retained enzymic activity on its natural substrate (Pol-CP) and was also capable of hydrolysing the synthetic peptide acyl-Ala-Ala-Val-Tyr-HisGln-Ala-Ala-NH,, derived from the consensus cleavage site for the TuMV polyprotein. Analysis by mass spectrometry of the two fragments resulting from this reaction indicated that cleavage took place between the Gln and Ala residues, as expected. A fluorogenic derivative of this peptide was hydrolysed by VPgPro, affording a convenient quantitative assay for intermolecular proteolytic activity, and was used to determine the pH-activity profile. The availability of large quantities of pure proteinase and of a rapid and sensitive assay will permit detailed kinetic and structural studies which are essential to obtain a better understanding of the mode of action of this and related viral proteinases, such as the 3C proteinase of picornaviruses.

The 49-kDa, nuclear inclusion a-like, viral protein genome-linked proteinase (VPg-Pro) of turnip mosaic potyvirus (TuMV) was expressed in Escherichia coli. The protein was produced in a soluble form at high levels and was active, as demonstrated by intermolecular cleavage of the polymerase capsid protein (Pol-CP) substrate. The VPg-Pro was purified by metal-chelation and ion-exchange chromatographies. Two forms of VPg-Pro, which differed in molecular masses, were obtained during isolation; their identities were confirmed by immunoblot analysis and N-terminal amino acid sequencing. Data indicated that cleavage took place at a site near the C-terminus of VPg-Pro and was the result of the proteolytic activity of the viral protein. The purified proteinase retained enzymic activity on its natural substrate (Pol-CP) and was also capable of hydrolysing the synthetic peptide acyl-Ala-Ala-Val-Tyr-HisGln-Ala-Ala-NH,, derived from the consensus cleavage site for the TuMV polyprotein. Analysis by mass spectrometry of the two fragments resulting from this reaction indicated that cleavage took place between the Gln and Ala residues, as expected. A fluorogenic derivative of this peptide was hydrolysed by VPgPro, affording a convenient quantitative assay for intermolecular proteolytic activity, and was used to determine the pH-activity profile. The availability of large quantities of pure proteinase and of a rapid and sensitive assay will permit detailed kinetic and structural studies which are essential to obtain a better understanding of the mode of action of this and related viral proteinases, such as the 3C proteinase of picornaviruses.

No abstract