An In Vivo Biocatalytic Cascade Featuring an Artificial Enzyme Catalyzed New-to-Nature Reaction

Artificial enzymes utilizing the genetically encoded non-proteinogenic amino acid p-aminophenylalanine (pAF) as catalytic residue are able to react with carbonyl compounds through an iminium ion mechanism, making reactions possible that have no equivalent in nature. Here, we report an in vivo biocatalytic cascade that is augmented with such an artificial enzyme catalyzed new-to-nature reaction. The artificial enzyme in this study is a pAF containing evolved variant of the Lactococcal multidrug resistance Regulator, designated LmrR_V15pAF_RMH, which efficiently converts in vivo produced benzaldehyde derivatives into the corresponding hydrazone products inside E. coli cells. These in vivo biocatalytic cascades comprising an artificial enzyme catalyzed reactions are an important step towards achieving a hybrid metabolism.

Tandem Friedel-Crafts-Alkylation-Enantioselective-Protonation by Artificial Enzyme Iminium Catalysis

The incorporation of organocatalysts into protein scaffolds, i.e. the production of organocatalytic artificial enzymes, holds the promise of overcoming some of the limitations of this powerful catalytic approach. In particular, transformations for which good reactivity or selectivity is challenging for organocatalysts may find particular benefit from translation into a protein scaffold so that its chiral microenvironment can be utilised in catalysis. Previously, we showed that incorporation of the non-canonical amino acid para-aminophenylalanine into the non-enzymatic protein scaffold LmrR forms a proficient and enantioselective artificial enzyme (LmrR_pAF) for the Friedel-Crafts alkylation of indoles with enals. The unnatural aniline side-chain is directly involved in catalysis, operating via a well-known organocatalytic iminium-based mechanism. In this study, we show that LmrR_pAF can enantioselectively form tertiary carbon centres not only during C-C bond formation, but also by enantioselective protonation. Control over this process is an ongoing challenge for small-molecule catalysts for which general solutions do not exist. LmrR_pAF can selectively deliver a proton to one face of a prochiral enamine intermediate delivering product enantiomeric excesses and yields that rival the best organocatalyst for this transformation. The importance of various side-chains in the pocket of LmrR is distinct from the Friedel-Crafts reaction without enantioselective protonation, and two particularly important residues were probed by exhaustive mutagenesis. This study shows how organocatalytic artificial enzymes can provide solutions to transformations which otherwise require empirical optimisation and design of multifunctional small molecule catalysts.

Evidence for an N-halohistidyl Intermediate in the Catalytic Cycle of Vanadium Chloroperoxidase (VCPO) and an Artificial Enzyme derived from VCPO: A Computational Investigation

Vanadium haloperoxidases play an important catalytic role in the natural production of antibiotics which are difficult to make in the laboratory. Understanding the catalytic mechanism of these enzymes will aide in the production of artificial enzymes useful in bioengineering the synthesis of drugs and useful chemicals. However, the catalytic mechanism remains not fully understood yet. In this paper, we investigate one of the key steps of the catalytic mechanism using QM/MM. Our investigation reveals a new N-haloxy histidyl intermediate in the catalytic cycle of vanadium chloroperoxidase (VCPO). This new intermediate, in turn, yields an explanation for the known inhibition of the enzyme by substrate under acidic conditions (pH<4). Additionally, we examine the possibility of replacing V in VCPO by Nb or Ta using QM modeling. We report the new result that the Gibbs free energy barrier of several steps of the catalytic cycle are lower in the case of artificial enzymes, incorporating NbO43- or TaO43- instead of VO43-. Our results suggest that these new artificial enzymes may catalyze the oxidation of halide faster than the natural enzyme.

Theoretical Studies of the Self Cleavage Pistol Ribozyme Mechanism

Ribozymes are huge complex biological catalysts composed of a combination of RNA and proteins. Nevertheless, there is a reduced number of small ribozymes, the self-cleavage ribozymes, that are formed just by RNA and, apparently, they existed in cells of primitive biological systems. Unveiling the details of these “fossils” enzymes can contribute not only to the understanding of the origins of life but also to the development of new simplified artificial enzymes. A computational study of the reactivity of the pistol ribozyme carried out by means of classical MD simulations and QM/MM hybrid calculations is herein presented to clarify its catalytic mechanism. Analysis of the geometries along independent MD simulations with different protonation states of the active site basic species reveals that only the canonical system, with no additional protonation changes, renders reactive conformations. A change in the coordination sphere of the Mg2+ ion has been observed during the simulations, which allows proposing a mechanism to explain the unique mode of action of the pistol ribozyme by comparison with other ribozymes. The present results are at the center of the debate originated from recent experimental and theoretical studies on pistol ribozyme.