In vivo promiscuity and directed evolution of oligomeric beta-decarboxylating dehydrogenases

At the molecular level, natural evolution gave rise a wide variety of proteins endowed with exquisite properties. In this study, we envisioned to contribute a better understanding of the evolutionary mechanisms and structure function relationships of oligomeric enzymes. Beta-decarboxylating dehydrogenases were chosen as a model superfamily. Oligomerization is an advantage for complexifying the properties of a protein such as affording allosterism; however, the impact of oligomerization on the evolutionary mechanisms of proteins is poorly documented. This study will explore the potential of β-decarboxylating dehydrogenases as a platform for directed evolution of oligomeric enzymes using a strategy of activity interconversion between the family members. We first studied the promiscuous activities of D-malate and 3-isopropylmalate dehydrogenases (DmlA and IPMDH) in vivo and showed reciprocal complementation of each enzyme in Escherichia coli strains where the other enzyme is absent. By simulating gene duplication, we demonstrated that heteromeric complex of DmlA is formed in vivo from the interaction of the subunits encoded from the two copies of the gene. Following a directed evolution approach, we successfully generated a NAD dependent E. coli isocitrate dehydrogenase (IDH) from DmlA. We found that a single amino acid substitution (Leu89Ser) endows DmlA with about 60000-fold improvement in IDH activity although the IDH phenotype in vivo is thermosensitive. Co-expression of the wild type DmlA enzyme with the mutant did not result in an increased thermostability indicating that, in this specific case of molecular evolution, there is no paralogous chaperoning from the parental protein in the oligomeric assembly.(SC - Sciences) -- UCL, 201

Promiscuous activity of 3-isopropylmalate dehydrogenase produced at physiological level affords Escherichia coli growth on d-malate.

Promiscuous activities of enzymes may serve as starting points for the evolution of new functions. However, most experimental examples of promiscuity affording an observable phenotype necessitate the artificial overexpression of the target enzyme. Here, we show that 3-isopropylmalate dehydrogenase (IPMDH), an enzyme involved in leucine biosynthesis, has a secondary activity on d-malate, which is sufficient for d-malate assimilation under physiological conditions where the enzyme is upregulated. In vitro, the turnover constant (k ) of IPMDH for d-malate is about 30-fold lower than the k for 3-isopropylmalate, yet sufficiently high to support the growth on d-malate. From an evolutionary perspective, our results highlight the possibility of phenotype emergence triggered by arbitrary changes in environmental conditions and prior to any mutational event

Escherichia coli D-malate dehydrogenase, a generalist enzyme active in the leucine biosynthesis pathway.

The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of D-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on D-malate as a carbon source, the D-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB(-) strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (L(+)-tartrate, D-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution

Flow mode enantioselective transamination using transaminase enzymes immobilized in a macroporous silica monolith

Here, we present the immobilization of TA on cylindrical silica monoliths that are particularly appropriate for the design of continuous flow reactors. The monoliths are prepared by a bottom-up sol-gel method based on emulsion templating. They minimize pressure drop, ensure a plug flow regime, and are easy to manipulate. Their surface is functionalized to bring epoxy or amino groups on the surface, allowing to anchor the enzyme. A simple flow reactor featuring covalently immobilized TA (ATA-117 from Codexis) is presented as a proof-of-concept. The transamination of pyruvate with racemic 4-bromo-α-methylbenzylamine (BMBA) to produce bromoacetophenone (BAP) was used as a model reaction (kinetic resolution). We show how the immobilization of the enzyme – and therefore the catalyst performance – can be optimized by tuning parameters of the monolith surface functionalization

Immobilizing transaminase in a macroporous silica monolith for flow mode enantioselective transamination

In this communication, we will present the immobilization of TA on silica monoliths that are particularly appropriate for the design of continuous flow reactors. The silica monoliths are prepared by a bottom-up sol-gel method based on emulsion templating. They minimize pressure drop, ensure a plug flow regime, and are easy to manipulate. After presenting the proof-of-concept with a simple flow reactor featuring covalently immobilized TA (ATA-117 from Codexis), we will show how the immobilization of the enzyme – and therefore the catalyst performance – can be optimized by tuning parameters of the monolith surface functionalization. We will show that a self-produced TA of higher purity allows reaching higher production rates. We will also disclose an attempt to run a chiral amine synthesis using a cascade biocatalytic process to shift the thermodynamic equilibrium of the reaction towards the desired product, as proposed by Turner et al., but here in flow mode

Methyl arachidonyl fluorophosphonate inhibits Mycobacterium tuberculosis thioesterase TesA and globally affects vancomycin susceptibility

International audiencePhthiocerol dimycocerosates and phenolic glycolipids (PGL) are considered as major virulence elements of Mycobacterium tuberculosis, in particular because of their involvement in cell wall impermeability and drug resistance. The biosynthesis of these waxy lipids involves multiple enzymes, including thioesterase A (TesA). We observed that purified recombinant M. tuberculosis TesA is able to dimerize in the presence of palmitoyl-CoA and our 3D structure model of TesA with this acyl-CoA suggests hydrophobic interaction requirement for dimerization. Furthermore, we identified that methyl arachi-donyl fluorophosphonate, which inhibits TesA by covalently modifying the catalytic serine, also displays a synergistic antimicrobial activity with van-comycin further warranting the development of TesA inhibitors as valuable antituberculous drug candidates