Establishing wild-type levels of catalytic activity on natural and artificial (βα) ₈ -barrel protein scaffolds

Abstract

The generation of high levels of new catalytic activities on natural and artificial protein scaffolds is a major goal of enzyme engineering. Here, we used random mutagenesis and selection in vivo to establish a sugar isomerisation reaction on both a natural (␤␣) 8-barrel enzyme and a catalytically inert chimeric (␤␣) 8-barrel scaffold, which was generated by the recombination of 2 (␤␣) 4-half barrels. The best evolved variants show turnover numbers and substrate affinities that are similar to those of wild-type enzymes catalyzing the same reaction. The determination of the crystal structure of the most proficient variant allowed us to model the substrate sugar in the novel active site and to elucidate the mechanistic basis of the newly established activity. The results demonstrate that natural and inert artificial protein scaffolds can be converted into highly proficient enzymes in the laboratory, and provide insights into the mechanisms of enzyme evolution.chimeric protein ͉ enzyme design ͉ enzyme evolution ͉ half barrel ͉ TIM barrel

Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis

“…This is in contrast to the bisubstrate enzyme PriA, in which the presence of an aspartate in the same position is tolerated. Our structural data on the PriA-PrFAR complex, by indicating direct involvement of Asp130 in catalysis, also explain why this replacement in the HisA variant with TrpFlike activity loses the original ProFAR isomerization activity (20).…”

“…A comparison of the structure of PriA and that of the directed evolution version of HisA (20) shows that two of the four mutated HisA residues (D127V, D169V) have equivalents (Asp130, Asp175) in the PriA active site with key roles for PriA catalysis, as evidenced by our structural and biochemical data. As mutation of Asp175 in PriA abolishes activity for both ProFAR and PRA isomerization while being required for PRA isomerization activity in HisA, the suggested role of the residue as an acid/base catalyst inevitably needs to be carried out by a different active site residue in the engineered HisA variant with TrpF-like activity, assuming a similar biochemical isomerization mechanism.…”

“…Therefore, ultimate proof of these observations will require further studies of bacterial species without a trpF operon gene under native living conditions. Nevertheless, irrespective of the precise evolutionary relationships among PriA, TrpF, and HisA enzymes, our data allow a mechanistic comparison with recent laboratory-based evolution experiments to convert the scaffold of the single-substrate substrate enzyme HisA from T. maritima into an enzyme with TrpF-like properties catalyzing the isomerization of PRA-albeit at the expense of its native ProFAR isomerization activity (15,20).…”

Metamorphic proteins mediate evolutionary transitions of structure

“…These structures also support the notion that β-propellers evolved via the multiplication of smaller gene segments (16) and could assemble with different numbers of such elements. In contrast, ðβ∕αÞ 8 -barrels are also thought to have evolved through a process of duplication and fusion of smaller units (4,27,28) but are known to exist with fourfold symmetry only (8 β/α modules). Two new β-propeller arrangements (and possibly many more) that are functional could be easily derived from the same starting protein.…”

“…This restriction imposes a major challenge, because mutational steps may create large structural perturbations that need to be tolerated while retaining function (2). In single domain proteins, significant "cut and paste" structural changes perturb a well-packed hydrophobic core (3,4). Evolutionary transitions from one folded and functional domain structure into a new one should therefore (or must, by default, if one follows Maynard-Smith's conjecture) involve intermediates able to adopt more than one structure (5,6).…”