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AphA is a magnesium-dependent, bacterial class B acid phosphatase that catalyzes the hydrolysis of a variety of phosphoester substrates and belongs to the DDDD superfamily of phosphohydrolases. The recently reported crystal structure of AphA from Escherichia coli has revealed the quaternary structure of the enzyme together with hints about its catalytic mechanism. The present work reports the crystal structures of AphA from E. coli in complex with substrate, transition-state, and intermediate analogues. The structures provide new insights into the mechanism of the enzyme and allow a revision of some aspects of the previously proposed mechanism that have broader implications for all the phosphatases of the DDDD superfamily.

The Escherichia coli gene aphA codes for a periplasmic acid phosphatase called AphA, belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. After our first report about its crystal structure, we have started a series of crystallographic studies aimed at understanding of the catalytic mechanism of the enzyme. Here, we report three crystal structures of the AphA enzyme in complex with the hydrolysis products of nucleoside monophosphate substrates and a fourth with a proposed intermediate analogue that appears to be covalently bound to the enzyme. Comparison with the native enzyme structure and with the available X-ray structures of different phosphatases provides clues about the enzyme chemistry and allows us to propose a catalytic mechanism for AphA, and to discuss it with respect to the mechanism of other bacterial and human phosphatases.

AphA is a periplasmic acid phosphatase of Escherichia coli belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. The crystal structure of AphA has been determined at 2.2 A and its resolution extended to 1.7 A on an AuCl 3 derivative. This represents the first crystal structure of a class B bacterial phosphatase. Despite the lack of sequence homology, the AphA structure reveals a haloacid dehalogenase-like fold. This finding suggests that this fold could be conserved among members of the DDDD superfamily of phosphohydrolases. The active enzyme is a homotetramer built by using an extended N-terminal arm intertwining the four monomers. The active site of the native enzyme, as prepared, hosts a magnesium ion, which can be replaced by other metal ions. The structure explains the non-specific behaviour of AphA towards substrates, while a structure-based alignment with other phosphatases provides clues about the catalytic mechanism.