Abstract
The high resolution crystal structure of D-amino-acid oxidase (DAAO) from the yeast Rhodotorula gracilis provided us with the tool to engineer the substrate specificity of this flavo-oxidase. DAAO catalyzes the oxidative deamination of D-amino acids, with the exception of D-aspartate and D-glutamate (which are oxidized by D-aspartate oxidase, DASPO). Following sequence homology, molecular modeling, and simulated annealing docking analyses, the active site residue Met-213 was mutated to arginine. The mutant enzyme showed properties close to those of DASPO (e.g. the oxidation of D-aspartate and the binding of l-tartrate), and it was still active on D-alanine. The presence of an additional guanidinium group in the active site of the DAAO mutant allowed the binding (and thus the oxidation) of D-aspartate, but it was also responsible for a lower catalytic activity on D-alanine. Similar results were also obtained when two additional arginines were simultaneously introduced in the active site of DAAO (M213R/Y238R mutant, yielding an architecture of the active site more similar to that obtained for the DASPO model), but the double mutant showed very low stability in solution. The decrease in maximal activity observed with these DAAO mutants could be due to alterations in the precise orbital alignment required for efficient catalysis, although even the change in the redox properties (more evident in the DAAO-benzoate complex) could play a role. The rational design approach was successful in producing an enzymatic activity with a new, broader substrate specificity, and this approach could also be used to develop DAAO variants suitable for use in biotechnological applications.
Highlights
D-Amino-acid oxidase (EC 1.4.3.3, D-amino-acid oxidase (DAAO))1 is considered the paradigm of the dehydrogenase-oxidase class of flavoproteins [1]
A comparison of the active site of DAAO with the model derived for DASPO showed the presence of two additional arginines in the active site of the latter, which are not present in RgDAAO (Fig. 1B); Arg-216 and Arg-237 seemed to be located in a position resembling that of Met-213 and Tyr-238 in DAAO
D-Aspartate binding into the active site of both RgDAAO and DASPO model was achieved using a Monte Carlo-simulated annealing algorithm that predicted the bound conformations of flexible ligands to macromolecules (AutoDock 2.4 software) [30, 31]
Summary
Site-directed Mutagenesis and Enzyme Expression—Enzymatic DNA modifications were carried out according to the manufacturer’s instructions and essentially as described in Sambrook et al [21]. Redox potentials of the M213R mutant were determined by the method of dye equilibration [24] using the xanthine/xanthine oxidase reduction system [25] at 15 °C and in the absence of EDTA and 2-mercaptoethanol. Small volumes (25–200 l) of concentrated D-amino acid solution were added to 5 ml of 100 mM sodium pyrophosphate buffer, pH 8.5, in the cell, while stirring at a constant rate and applying a fixed voltage (ϩ400 mV versus Ag/AgCl). The steady-state anodic currents were recorded ϳ3 min after a fixed amount of enzyme had been added in the measuring cell (the time required to reach 95% of the steady-state anodic current) It was demonstrated for wild-type DAAO that the slope of the current intensity increase, as well as the finale value reached, depends on the D-amino acid concentration [20]. Structure Coordinates of RgDAAO—In complex with trifluoro-D-alanine, the Protein Data Bank code is 1c0p [7]; in complex with anthranilate the Protein Data Bank accession code is 1c0i
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