Narrowing Substrate Specificity in a Directly Evolved Enzyme:  The A293D Mutant of Aspartate Aminotransferase^,

Abstract

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.

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

“…It is therefore possible that small movements of the Nt-tail might be able to affect the activity of the opposite subunit. Also, the crystal structures of eAATase 12,37 and our stability studies 14 show that the interface between the two large domains is formed by a large interconnected network of electrostatic interactions and hydrogen bonds. This might mediate the weak allosteric effects observed in this study.…”

“…Mal and Hca are selective competitive inhibitors of eAATase 7 and SRHEPT þ A293D, 12 respectively (Table III, last four rows). In the OCT/WT context, Mal is expected to bind preferentially to the AATase site and Hca to the w-TATase site (Scheme 1).…”

“…The HEX variant, which does not complement the E. coli TATase activity in vivo, was improved by directed evolution into an enzyme (HEX þ A293D) that complements that function with only the single additional mutation. 9 Moreover, A293D enhances the specificities of HEX 9 and SRHEPT 12 ; it makes them more TATase like. SRHEPT þ A293D is a very efficient enzyme whose substrate specificity is within the range of those observed for natural TATases 13 (Kathryn Muratore, PhD thesis, UC Berkeley), and it is very similar to that of eTATase.…”

“…SRHEPT þ A293D is a very efficient enzyme whose substrate specificity is within the range of those observed for natural TATases 13 (Kathryn Muratore, PhD thesis, UC Berkeley), and it is very similar to that of eTATase. 12 The accumulated knowledge on aminotransferase specificity was used here to design an enzyme in which one active site would behave as an AATase and the other as a TATase. The option of using natural AATase and TATase monomers as the constituents of this hybrid enzyme was discarded due to structural constraints: first, although most of the active site is nested within one monomer, some essential residues are provided by the opposite one.…”

“…For example, if each binding pocket in the heterodimer retains the same specificity as it has in the respective homodimeric context, then each should preferentially associate with a specific competitive inhibitor: maleate (Mal) in the eAATase active site 7 and hydrocinnamate (Hca) in the SRHEPTþA293D active site (Scheme 1). 12 In that asymmetric scenario, long range communication between active sites should be easier to detect than in a homodimeric context, where it Scheme 1. Substrate specificity and inhibitor selectivity of OCT/WT.…”