Fig 1.
Principle of the end-point alkali assay method for the high-throughput screening of kinase activity.
Fig 2.
(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.
Fig 3.
High-throughput method used to screen LysC mutants active on malate.
(A) Procedure for the identification and isolation of recombinant malate kinase enzyme from generated clones. (B) Screening method validation strategy. (C) LysC activities on the natural substrate aspartate (positive control) and the non-natural substrate (L)-malate (negative control) determined for a single microplate. Activities (black circles) are plotted in descending order. Mean activities and their standard deviations are shown as solid and dotted horizontal black lines, respectively.
Fig 4.
Malate kinase activities of the nine positive clones identified by screening of crude cell extracts.
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.
Table 1.
Kinetic parameters on (L)-malate and (L)-aspartate for LysC wild-type and mutants.
Table 2.
Utilisation of additional (L)-malate binding energy gained by LysC mutation in stabilising enzyme-substrate and enzyme transition-state complexes.
Fig 5.
Enzyme binding site interactions in a modelled complex of the LysC E119S:V115A double mutant with (L)-malate and Mg-ADP.
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 holo 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 apo 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.
Fig 6.
Molecular model of a ternary complex of the V115A, E119S Lys-C mutant with (L)-malate and Mg-ADP.
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.
Table 3.
Calculated (L)-malate electrostatic binding interaction energies in the V115A:E119S and V115A:E119S:E434V Ec-LysC mutant ternary complexes with Mg-ADP.