Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

Abstract

The promiscuous activity of the enzyme o-Succinylbenzoate Synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both Density Functional Theory and Semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylaminoacids, being N-succinyl-Rphenylglycine the best substrate. We investigated the molecular mechanisms for both reactions exploring the Potential Energy Surface. Then, Molecular Dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues with the reacting system. Our results confirm the plausibility of the reaction mechanisms proposed in the literature, with a good agreement between theoretical and experimentally-derived activation free energies. Our simulations unravel the role played by the different residues in each of the two possible reactions. The presence of flexible loops in the active site and the selection of structural modifications in the substrate seem to be key elements to promote the promiscuity of this enzyme.

New light on bacterial carbonic anhydrases phylogeny based on the analysis of signal peptide sequences

“…In fact, although the g class is widely distributed among all three domains of life, it is the only CA class mainly identified in Archaea, the most ancient microorganisms that exist on earth. The phylogenetic analysis is corroborated by the promiscuity theory, which is a key factor in the evolution of a new protein function 40,41 . Looking at the substrate used by g-, b-and a-CAs, we noticed that g-CAs use only CO 2 as substrate, b-CAs can hydrolyze CO 2 , COS and CS 2 , whereas the a-CAs not only hydrate CO 2 , CS 2, COS, cyanamide and cyanate, but also possess esterase activity, with a range of esters of carboxylic, sulfonic or phosphate esters 27,32,[42][43][44][45][46][47] .…”

N-succinylamino acid racemases: Enzymatic properties and biotechnological applications

“…These differences could be explained by the equilibrium model: enzymatic activity might be lowered or lost below the apparent unfolding temperature as a result of temperature-induced conformational changes at the active site from an optimum configuration for substrate binding to a less optimum one [81,82]. This conjecture is in agreement with the expected flexibility of a promiscuous active site, and is further supported by quantum mechanics/molecular mechanics (QM/MM) analysis in the case of NSAR [83]. Besides the "natural" NSAs racemization, the activity towards different NAAs, NCAs, NChAs, NBtAs, NPrAs, NBzAs or NFAs has been shown ( Table 3).…”

“…Mutation of two surface-exposed residues (Arg20 and Ser22) thought to assist in 20s loop closure through interaction with the cap domain, also resulted in decreased K M values, although their k cat were only slightly affected [71]. Finally, QM/MM analysis also supports the importance of this loop in AmyNSAR by providing similar interactions to different substrates, which can accommodate in a slight different orientation into the active site ( Figure 6) [83].…”

“…In the opposite direction of the reaction, Lys263 would behave as the base, and Lys163 as the acid. QM/MM studies support this mechanism[83]. Whereas the catalytic framework is highly conserved in the whole enolase superfamily[77], comparison of structural elements related to the activity of the different members of this superfamily, including the 20s and 50s loops, revealed a lower conservation.…”

Computer simulations of the catalytic mechanism of wild-type and mutant β-phosphoglucomutase

“…In order to verify that the substrate positioning after equilibration is not an artifact of having manually placed the substrate in the active site, we also performed molecular dynamics simulations of β-PGM in complex with a phosphonate analogue of β-G1P (PDB ID 4C4R, converting the phosphonate back to the phosphate by atom replacement) 13 to verify that the substrates equilibrate to similar positions in both cases. To reduce computational cost, we divided the system into three regions as is commonly performed in similar work: 24,25 a reactive region, containing all the reacting atoms taking part in the EVB calculation, an active region, where all the atoms within 24 Å of the transferring phosphorus atom were allowed to move freely, and an external region, where all the remaining atoms were constrained to their initial positions. The protonation states for all histidines were inspected using MolProbity, 26 as well as visual inspection, and protonation patterns (at the δvs.…”