Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme

Abstract

The leader proteinase (Lpro) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp163, which orients the catalytic histidine residue, and Asp164; the equivalent residues in papain are Asn175 and Ser176. The importance of these residues for Lpro activity was examined by site-directed mutagenesis. Replacement of Asp163 with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of Lpro, eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp164 by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp164, and to a lesser extent that of Asp163, is extremely important for Lpro to attain full activity upon synthesis.

Foot-and-Mouth Disease Virus Leader Proteinase: Specificity at the P2 and P3 Positions and Comparison with Other Papain-like Enzymes

“…Quantitation by densitometry of the amounts of the uncleaved Lb pro VP4VP2 precursor and mature Lb pro shows that at 12 min, self-processing had reached a level of 83% (Table 3). This is very similar to values previously reported for in Vitro translation using RRLs (8,16). The 12 min time point was chosen as the optimal point for comparing enzymatic activities because earlier time points cannot be measured with any accuracy.…”

Comparative complete genome analysis of Indian type A foot-and-mouth disease virus field isolates

“…The residues that form catalytic triad which includes C 51 , H 148 , and D 163 [27], residues involved in substrate recognition C 133 [28], residues implicated in autocatalysis E 76 and cleavage of initiation factor eIF4G H 109 and H 138 [29] and residues modulates the electrostatic charge at the catalytic site D 164 [30] were fully conserved in all the isolates reiterating the significance of these residues in functional activity of L pro . [32] was totally conserved.…”

Investigating the Substrate Specificity and Oligomerisation of the Leader Protease of Foot and Mouth Disease Virus using NMR

“…The tightness of the dimer is probably due to the many electrostatic interactions present between the active site and the CTE. 16 Third, we examined the stability of the dimer by size-exclusion chromatography. Lb pro eluted at a position corresponding to a dimer, whereas sLb pro eluted at a volume slightly higher than expected for a monomer.…”

“…Furthermore, attempts to investigate this region by sitedirected mutagenesis were unsuccessful. 16 To provide answers to the open questions of Lb pro self-processing, its oligomerization state in solution and the nature of its S1 site, we decided to study Lb pro and sLb pro by NMR, using the full repertoire of techniques available for studying proteins.…”