Several mutant Escherichia coli aspartate aminotransferases (eAATases) have been characterized in the attempt to evolve or rationally redesign the substrate specificity of eAATase into that of E. coli tyrosine aminotransferase (eTATase). These include HEX (designed), HEX + A293D (design followed by directed evolution), and SRHEPT (directed evolution). The A293D mutation realized from directed evolution of HEX is here imported into the SRHEPT platform by site-directed mutagenesis, resulting in an enzyme (SRHEPT + A293D) with nearly the same ratio of k(cat)/K(m)(Phe) to k(cat)/K(m)(Asp) as that of wild-type eTATase. The A293D substitution is an important specificity determinant; it selectively disfavors interactions with dicarboxylic substrates and inhibitors compared to aromatic ones. Context dependence analysis is generalized to provide quantitative comparisons of a common substitution in two or more different protein scaffolds. High-resolution crystal structures of ligand complexes of HEX + A293D, SRHEPT, and SRHEPT + A293D were determined. We find that in both SRHEPT + A293D and HEX + A293D, the additional mutation holds the Arg 292 side chain away from the active site to allow increased specificity for phenylalanine over aspartate. The resulting movement of Arg 292 allows greater flexibility of the small domain in HEX + A293D. While HEX is always in the closed conformation, HEX + A293D is observed in both the closed and a novel open conformation, allowing for more rapid product release.

The distribution of dust emitted by the supergiant star NML Cygni has been resolved by interferometry at 11 km wavelengths at various times over a period of 6 yr. Results show there are two discrete dust shells, which have both moved away from the star approximately the same amount during the 6 yr period. This allows determination of the time between ejection of material forming the two shells to be 65^14 yr. Assuming the radial outÑow velocity can be derived from Doppler-measured velocities of masers surrounding the star, its distance can be calculated from the observed angular motion to be 1220^300 pc. This decreases the luminosity of the star by about 1 mag over that deduced from the distance 1900 pc previously assumed.

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