The use of the periplasmic fraction as starting
The use of the periplasmic fraction as starting material and the presence of a unique purification step concurred in improving the purification yield to 25.3%, which is significantly higher than what obtained with the previous purification protocol (6.7%). In addition, the analysis of the kinetic constants evaluated on pNPR suggested that the His-tag did not significantly alter the α-RHA activity of RHA-Phis on this synthetic substrate. To gather more insights into the catalytic mechanism of RHA-Phis for a future fine-tuning of this biotechnologically relevant enzyme, a combined approach of sequence alignment and homology modeling was used. The alignment of RHA-P sequence with 130 homologs of the GH106 family allowed identifying five highly conserved Glu and Asp residues. To confirm the role of these conserved residues in the active site, the homology model of RHA-Phis was built by using the three-dimensional structure α-RHA BT0986 from Bacteroides thetaiotaomicron as template. The modeled structure shows that, whereas residues D503, E506 and E644 are conserved also in the template protein BT0986, positions D552 and D763 do not seem to be directly involved in the “architecture” of the active site. To shed light on this aspect, an alanine scanning strategy on all five conserved residues initially retrieved by the sequence alignment was performed. Both whole recombinant mtor inhibitor and purified proteins enzymatic assays show that mutants at positions D552 and D763 have residual rhamnosidase activity (Fig. 4). The different values of specific activity measured on pNPR for each mutant in the two different assays probably account for different expression levels of the mutant proteins in the strain BL21(DE3) of E. coli. Of course, this aspect becomes irrelevant when purified proteins are instead assayed for their rhamnosidase activity. Although the whole cells assay at this stage is undoubtedly less informative to evaluate the differential catalytic role of the mutagenized residues, its feasibility shows that we have an invaluable tool to perform in the near future the preliminary screening of a large number of RHA mutants. In addition, from a biotechnological point of view, the low stability of purified enzymes often favors the use of whole cells in the set-up and development of bioconversion processes. Purified D552 and D763 mutants exhibited a residual activity of 47.9% and 15.7%, respectively; likely, these residues are not directly involved in catalysis. This hypothesis is also supported by the kinetic constants measured on pNPR for these two mutants. Indeed, the decrease in catalysis efficiency, expressed by the k/KM, is almost exclusively dependent on the kcat value and not on a difference in the apparent affinity of the enzyme for the substrate. These positions might not be directly involved in the binding or positioning of the substrate in the active site, as confirmed by the RHA-Phis model described in this work, which confirms that these two residues are located in quite distant positions from the active site. The reduced specific activity observed in these mutants might thus be related to protein structural modifications leading to a soluble but partially inactive protein. More specifically, a possible functional role in the protein folding process can be foreseen for D763, for which a notable decrease in kcat value is observed compared to wt RHA-Phis. Residues D503, E506 and E644, which are conserved also in the template α-RHA BT0986, probably share a fundamental role in the active site of the enzyme. In particular, E506 is homologous to residue E461 in BT0986 (mutated in Q461 in the reference structure 5MWK) and represents the potential acid or base catalyst (highlighted in cyan in Fig. S3). On the other hand, residues D503 and E644 are homologous to residues D458 and E592 in BT0986, and seem to serve as calcium binding residues at the active site (these residues are highlighted in red in Fig. S3). The marked decrease of the activity observed when these residues are mutagenized supports the presence of an essential calcium ion at the active site of RHA-Phis as well, and a similar catalytic mechanism. It is worth noting that all four residues involved in the calcium binding in RHA-Phis model are conserved also in the template, thus indicating the importance of the calcium ion stabilization for the catalytic mechanism of the enzyme.