FIG 1 .

Enzymatic and structural comparisons of wild-type and mutant ADIs. (A) Model of the GAS ADI crystal structure illustrating the pseudo-5-fold axis of the catalytic domain in green, blue, pink, cyan, and red, respectively, with the helical orthogonal bundle in orange and the remaining structural elements in gray. (B) ADI active-site residues targeted for site-directed mutagenesis. (C) Topology diagram of GAS ADI, colored as in panel A. (D) Colorimetric assay for the determination of citrulline. Triplicate data from two independent replicate experiments are presented. Error bars indicate the standard deviations. Wild-type ADI produced a significantly greater amount of citrulline than each mutant ADI, as determined by one-way ANOVA (P < 0.05; indicated by an asterisk). (E) Tryptophan emission fluorescence spectra of wild-type ADI and mutant proteins. Wild-type ADI and the D166A, E220A, H275A, and D277A mutant ADIs exhibited nearly identical emission maxima (347 to 349 nm), whereas that of C401A mutant ADI was red shifted to 330 nm, which is indicative of significantly altered tertiary and/or quaternary structure. (F) UV CD spectra (200 to 260 nm) comparing the secondary structures of mutant ADIs to that of wild-type ADI. (G) ESI-MS spectra of wild-type and mutant ADIs. Wild-type ADI and the D166A, E220A, H275A, and D277A mutant ADIs exist as a monomer-dimer mixture, while C401A exists exclusively in a tetrameric form.