Glycine Oxidase Based High-Throughput Solid-Phase Assay for Substrate Profiling and Directed Evolution of (<i>R</i>)- and (<i>S</i>)‑Selective Amine Transaminases

Transaminases represent one of the most important enzymes of the biocatalytic toolbox for chiral amine synthesis as they allow asymmetric synthesis with quantitative yields and high enantioselectivity. In order to enable substrate profiling of transaminases for acceptance of different amines, a glycine oxidase and horseradish peroxidase coupled assay was developed. Transaminase activity is detected upon transfer of an amine group from an amino donor substrate to glyoxylate, generating glycine, which is subsequently oxidized by glycine oxidase, releasing hydrogen peroxide in turn. Horseradish peroxidase uses the hydrogen peroxide to produce benzoquinone, which forms a red quinone imine dye by a subsequent condensation reaction. As glycine does not carry a chiral center, both (<i>R</i>)- and (<i>S</i>)-selective transaminases accepting glyoxylate as amino acceptor are amenable to screening. The principle has been transferred to establish a high-throughput solid-phase assay which dramatically decreases the screening effort in directed evolution of transaminases, as only active variants are selected for further analysis

DataSheet1_Effect of Graphite Oxide on the Catalytic Behavior of (S)-Selective Amine Transaminases.PDF

Graphite oxide (GO) has been used for the immobilization of several classes of enzymes, exhibiting very interesting properties as an immobilization matrix. However, the effect the nanomaterial has on the enzyme cannot be predicted. Herein, the effect GO has on the catalytic behavior of several (S)-selective amine transaminases [(S)-ATAs] has been investigated. These enzymes were the focus of this work as they are homodimers with pyridoxal 5′-phosphate in their active site, significantly more complex systems than other enzymes previously studied. Addition of GO (up to 0.1 mg/ml) in the reaction medium leads to activation (up to 50% improved activity) for most enzymes studied, while they maintain their temperature profile (they perform better between 40 and 45°C) and their stability. However, the effect is not universal and there are enzymes that are negatively influenced by the presence of the nanomaterial. More profound is the effect on the (S)-ATA from Chromobacterium violaceum which loses almost 50% of its activity in the presence of 0.1 mg/ml GO, while the stability was significantly decreased, losing its activity after 2 h incubation at 40°C, in the presence of 25 μg/ml GO. This negative effect seems to rise from minor secondary structure alterations; namely, a loss of α-helices and subsequent increase in random coil (∼3% in the presence of 25 μg/ml GO). We hypothesize that the effect the GO has on (S)-ATAs is correlated to the surface chemistry of the enzymes; the less negatively-charged enzymes are deactivated from the interaction with GO. This insight will aid the rationalization of ATA immobilization onto carbon-based nanomaterials.</p

Revealing Cutinases’ Capabilities as Enantioselective Catalysts

The specific activity and enantioselectivity of immobilized cutinases from Humicola insolens (HiC) and Aspergillus oryzae (AoC) were compared with those of Lipase B from Candida antarctica (CALB) for a series of 1-phenylethanol (1-PEA) structural analogues. The aim was to understand their catalytic behavior by rationally studying three structural elements of the substrates: the length of the alkyl chain, the position of methylation of the aromatic ring, and the aromatic character of the ring. All enzymes were immobilized on the macroporous support Lewatit VP OC 1600 at loadings of ∼10% w/w. Docking studies revealed structural features of the enzymes that led to activity differences. All three enzymes exhibit (<i>R</i>)-selectivity. AoC, due to its more open and accessible active site, possesses high activity that exceeds in most cases that of HiC and CALB. By increasing the substrate’s alkyl chain length from methyl to <i>n</i>-propyl, the activity for the (<i>R</i>)-enantiomer of all three enzymes decreased significantly (≥70%), while the enantioselectivity of both cutinases was larger than that of CALB for the bulkier substrate. Methylation of the ring in the <i>ortho</i>-position led to loss of activity (≥55%); however, AoC retained substantial activity. For all three enzymes, the planar character of the substrate phenyl ring is crucial for stabilizing the substrate in the active sites via π–π stacking. HiC displays high enantioselectivity with most substrates, despite its wide active site, due to a “bottleneck” produced over the catalytic serine from Leu66 and Ile169

Data_Sheet_1.pdf

<p>S-adenosyl-L-homocysteine (SAH) hydrolases (SAHases) are involved in the regulation of methylation reactions in many organisms and are thus crucial for numerous cellular functions. Consequently, their dysregulation is associated with severe health problems. The SAHase-catalyzed reaction is reversible and both directions depend on the redox activity of nicotinamide adenine dinucleotide (NAD⁺) as a cofactor. Therefore, nicotinamide cofactor biomimetics (NCB) are a promising tool to modulate SAHase activity. In the present in vitro study, we investigated 10 synthetic truncated NAD⁺ analogs against a SAHase from the root-nodulating bacterium Bradyrhizobium elkanii. Among this set of analogs, one was identified to inhibit the SAHase in both directions. Isothermal titration calorimetry (ITC) and crystallography experiments suggest that the inhibitory effect is not mediated by a direct interaction with the protein. Neither the apo-enzyme (i.e., deprived of the natural cofactor), nor the holo-enzyme (i.e., in the NAD⁺-bound state) were found to bind the inhibitor. Yet, enzyme kinetics point to a non-competitive inhibition mechanism, where the inhibitor acts on both, the enzyme and enzyme-SAH complex. Based on our experimental results, we hypothesize that the NCB inhibits the enzyme via oxidation of the enzyme-bound NADH, which may be accessible through an open molecular gate, leaving the enzyme stalled in a configuration with oxidized cofactor, where the reaction intermediate can be neither converted nor released. Since the reaction mechanism of SAHase is quite uncommon, this kind of inhibition could be a viable pharmacological route, with a low risk of off-target effects. The NCB presented in this work could be used as a template for the development of more potent SAHase inhibitors.</p