Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox

Abstract

Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability.

Sac Pox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase

“…PLLs from extremophilic crenarchaeaon sources [3,4,16,34]. These counterparts exhibit industry-compatible properties ( e.g.…”

Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications

“…Although its natural robustness confers outstanding biotechnological potential on Sso Pox47, its catalytic activities were first increased to turn the biocatalyst into a cost-effective technology48. The resolution of the 3D-structure allowed the identification of the crucial role of residue W263 in both activity and substrate promiscuity15.…”

The Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis in Bacterial Metalloenzymes

“…[33] Further work has identified how essential the flexibility of the loops in PLL is to facilitate the promiscuous PTE activity. [144,149] In accordance with this, it has also been identified that loop conformations and flexibility are tuned through both directed and natural evolution. [142,145] Conformational substates occur throughout an enzyme-catalysed reaction, as product binding and release may require a more 'open' substate, and the chemical reaction itself will often require a 'closed' preorganised substate, as is observed in PTE.…”

“…This has been observed extensively in the natural, designed, and directed evolution towards enhanced organophosphate hydrolysis in proteins from several folds. [33,111,112,[142][143][144][145][146] In the evolution of PTE from the amidohydrolase superfamily, its latent lactonase activity and the promiscuity of PTE-like lactonases (PLLs) from different sources with organophosphate compounds indicated a likely evolutionary trajectory of PTE from PLLs, despite low sequence similarity (,30 %). [147][148][149] When analysing the sequence differences between PTEs and PLLs, the majority of the insertions, deletions and amino acid substitutions are located in the loop regions (Fig.…”