STRUCTURE AND FUNCTION OF PEPA AS AN AMINOPEPTIDASE AND IN XER SITE-SPECIFIC RECOMBINATION
Norbert Sträter1, S. D. Colloms2, D. J. Sherratt2
1Institut für Kristallographie, Freie
Universität Berlin, Takustr. 6, D-14195 Berlin, Germany
2Microbiology Unit, Department of
Biochemistry, University of Oxford, South Parks Road, Oxford OX1
3QU, UK
Aminopeptidase A from E. coli (PepA) is a zinc-containing aminopeptidase [1] which has an independent function as a DNA-binding protein in Xer site-specific recombination in a complex with three other proteins (XerC, XerD, ArgR) [2-5]. The Xer system acts at sites found in multicopy plasmids to monomerize plasmid multimers. Xer recombination also acts at dif, in the replication terminus region of the E. coli chromosome, to insure faithful segregation of newly replicated chromosomes to daughter cells at cell division. PepA is a main factor in DNA binding and recognition besides ArgR. A model for the Xer complex has been proposed in which the DNA wraps around PepA and ArgR [4].
PepA is a homohexameric protein of 55 kDa subunit molecular mass and is related to leucine aminopeptidase, which contains two zinc ions in the active site. Several crystal structures of leucine aminopeptidase from bovine lens are available with and without transition-state analogue inhibitors, which allowed a detailed analysis of the enzyme mechanism [6-9]. However, the bovine enzyme has no known DNA binding function. The two proteins have 31 % sequence identity (52 % in the catalytic C-terminal domain).
Crystals of wild-type PepA have been obtained by low-salt precipitation in space group P3 with cell dimensions of a = 177.1 A and c = 244.8 A diffracting to at least 2.7 A. A large number of subunits in the asymmetric unit (probably 10 - 12) made a structure solution by molecular replacement using the model of leucine aminopeptidase impossible. No other crystal form could be found for the wild-type enzyme. Current efforts focus on a structure solution by heavy atom derivatives and the anomalous scattering of the dizinc site. We also prepared mutant proteins by site-directed mutagenesis of surface residues, including the N-terminal and C-terminal region, in order to obtain a different crystal form. Out of 10 mutants which have been prepared for this purpose two new crystal forms were found very recently. One of these is suitable for a crystallographic structure determination and might enable a structure solution by molecular replacement. This rhombohedral crystal form (space group R32) has cell dimensions a = 198.3 A and c = 116.4 A.
Our interest in structural studies on PepA focuses on the mechanism of peptide hydrolysis by the dizinc active site as well as on the independent role of the protein in Xer site-specific recombination.