Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure

Previously, we determined the DNA and amino acid sequences as well as bioch emical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalyti c properties differed widely in terms of specific activity, substrate speci ficity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the prese nt investigation, to increase the specific activity of Aspergillus fumigatu s phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase kno wn at 2.5 Angstrom resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconser ved active site residues that might correlate with a given favorable proper ty of interest. Using this approach, Gln27 of A. fumigatus phytase (amino a cid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsi ble for the considerably lower specific activity (26.5 vs. 196 U . [mg prot ein](-1) at pH 5.0) of A. fumigatus phytase when compared to Aspergillus te rreus phytase, which has a Leu at the equivalent position. Site-directed mu tagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the spe cific activity to 92.1 U . (mg protein)(-1), and this and other mutations a t position 27 yielded an interesting array of pH activity profiles and subs trate specificities. Analysis of computer models of enzyme-substrate comple xes suggested that Gln27 of wild-type A. fumigatus phytase forms a hydrogen bond with the 6-phosphate group of myo-inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydroge n bond were indeed responsible for the differences in specific activity, th is would suggest product release as the rate-limiting step of the A. fumiga tus wild-type phytase reaction.