A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase

<div><p>An end-point ADP/NAD⁺ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD⁺ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD⁺ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (<i>k</i>_cat/<i>K</i>_m)_malate/(<i>k</i>_cat/<i>K</i>_m)_aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site.</p></div

Principle of the end-point alkali assay method for the high-throughput screening of kinase activity.

<p>Principle of the end-point alkali assay method for the high-throughput screening of kinase activity.</p

Calculated (L)-malate electrostatic binding interaction energies in the V115A:E119S and V115A:E119S:E434V Ec-LysC mutant ternary complexes with Mg-ADP.

<p>Calculated (L)-malate electrostatic binding interaction energies in the V115A:E119S and V115A:E119S:E434V Ec-LysC mutant ternary complexes with Mg-ADP.</p

Screening assay set-up.

<p>(A) Absorbance spectra of NADH and NAD⁺ co-enzyme solutions, at an initial concentration of 0.5 mM, during the sequential acid/alkali treatment. UV-visible spectra were recorded on solutions of the pure co-enzyme forms (blue curves), after acid (red curves) and alkali treatment (green curves). (B) Absorbance at 360 nm, determined in microplate format, as a function of NAD⁺ alkali derivative concentration assuming the total conversion of the NAD⁺ co-enzyme initially present. (C) NAD⁺ alkali derivative calibration curve, determined in microplate format, using solutions mimicking a reaction medium, typically containing inactivated enzyme crude extract, aspartate, ATP and a varying NADH/NAD⁺ co-enzyme ratio at a fixed final total concentration of the reduced and oxidised forms of 1.5 mM.</p

Utilisation of additional (L)-malate binding energy gained by LysC mutation in stabilising enzyme-substrate and enzyme transition-state complexes.

<p>Utilisation of additional (L)-malate binding energy gained by LysC mutation in stabilising enzyme-substrate and enzyme transition-state complexes.</p

Enzyme binding site interactions in a modelled complex of the LysC E119S:V115A double mutant with (L)-malate and Mg-ADP.

<p>A network of direct and water-mediated interactions between (L)-malate and enzyme residues and Mg-ADP is depicted as dashed line orange vectors connecting donor and acceptor heavy-atom co-ordinate positions. The Mg²⁺ ion is shown as an ochre-coloured space filling sphere, and water molecules mediating substrate binding and metal ion co-ordination interactions as cyan-coloured spheres. Atoms in (thick) stick representations of (L)-malate, ADP and labelled mutant enzyme side-chains are coloured according to element type: carbon, grey; nitrogen, blue; oxygen, red; and phosphorus, orange. The oxygen atom of the 2-OH hydroxyl group of (L)-malate that replaces the charged α-NH3 group in (L)-aspartate is indicated. Carbon atoms in (thin) stick side-chains representations in an overlay of the X-ray structure of the R-state <i>holo</i> complex of the wild-type enzyme with (L)-aspartate and Mg-ADP (PDB code 2j0w) are shown in green. For comparison an alternative E119 side-chain conformation observed in the inactive T-state <i>apo</i> form of the wild type enzyme (PDB code 2j0x), re-constructed on the 2j0w backbone, is depicted in (thin) stick representation with yellow-coloured carbon atoms.</p

Molecular model of a ternary complex of the V115A, E119S Lys-C mutant with (L)-malate and Mg-ADP.

<p>Cartoon representations of the dimer subunits are coloured grey and green. (L)-malate and Mg- ADP are shown as van der Waals spheres coloured according to element type: carbon, grey; nitrogen, blue; oxygen, red; phosphorus, orange; magnesium, ochre Side-chain atoms in the E434 residues at the enzyme surface are highlighted as orange-coloured van der Waals spheres. E434 lies approximately 27Å from the (L)-malate substrate molecule bound in one of the two active sites.</p

Malate kinase activities of the nine positive clones identified by screening of crude cell extracts.

<p>A control comparison is made with a clone expressing the wild-type enzyme (WT), corresponding to non-specific and/or malate-dependent endogenous NADH-dependent oxidoreductase enzymes.</p