X-ray structures of DAP D-aminopeptidase mutants and their penicillin complexes

J. Stout, S. N. Savvides, J.J. Van Beeumen

Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium

The Ochrobactrum anthropi D-aminopeptidase [DAP] can be considered a penicillin-binding protein (PBP) on the basis of its primary structure and the fact that it is inhibited by β-lactam compounds. It is a three domain enzyme of which the N-terminal, catalytic domain (domain A) has striking structural similarity to Streptomyces R61 D,D-carboxypeptidase (R61) and Class A and C β-lactamases [1, 2].

All these enzymes rely on an acyl-serine based reaction mechanism. In fact, their active site structures are quite similar [3]. Thorough study of these known structures revealed that rather subtle differences are responsible for their carboxypeptidase, aminopeptidase or β-lactamase activity respectively. Concerning the carboxy-peptidases it is shown that the substrate binding site is positively charged. Thus specific recognition of the C-terminus of the peptide substrate is possible. In contrast, the substrate binding site of DAP contains (only) one charged residue, Asp481, that might be responsible for the specific recognition of the N-terminus of the substrate. This residue is part of an 11 amino acid loop (the 476-486 loop or γ-loop) that protrudes into the active site (Figure 1). Enzymatic studies with DAP mutants, in which the γ-loop is deleted by directed mutagenesis, showed that this loop, which is absent from the related enzymes, determined the aminopeptidase activity of DAP (unpublished results).

Comparison of the Class C β-lactamase structures with the DAP structure showed that all proteins have an arginine residue in the same location in the active site. This residue has been proposed to participate in the binding of the carboxylic acid of β-lactams. Furthermore analysis of the distances separating the Oγ atom of the catalytic serine and atoms of the surrounding conserved residues made clear that the F distance (the distance that spans the oxyanion pocket of the active site) is wider Class A β-lactamases and DAP than in other PBP’s. This allows a better binding of the β-lactams in the active site in Class A β-lactamases. In the structure of DAP the F distance is also increased, however DAP preferentially binds DD-peptides over β-lactams. This is due to the presence of the γ-loop in the active site of DAP.

The comparison of the topology and the catalytic site geometry of domain A of DAP with that of a DD-carboxypeptidase and class C and A β-lactamases confirms the broad anatomical similarities suggested by sequence comparisons. Compared to the latter, the different substrate- and β-lactam-specifities are probably due to the presence of the γ-loop of domain C protruding into the active site

of the enzyme. Therefore, it is of considerable interest to study the structures of mutant DAP in which this loop is deleted. Two recombinant DAP mutants are available to us: DAP 475G487 and DAP475G487 N275R. In these mutants the γ-loop is specifically deleted, apart from that Asn275 is mutated into an arginine in the latter mutant. This residue is believed to be necessary for the specific recognition of the substrate carboxylate in closely related carboxypeptidases. It is shown that both mutants not only lose their D-aminopeptidase activity drastically, furthermore they become

more sensitive to substituted β-lactam compounds. In fact the mutants now invert towards a D,D-carboxypeptidase activity, especially when Asn275 is mutated into a basic residue. We wish to determine the X-ray structures of both the uncomplexed mutant enzyme and the mutant enzyme in

complex with β-lactam compounds. Penicillin G and 6-amino-penicillinic acid (6-APA) form stable acyl-enzyme complexes with this DAP mutant and will be used for structure determination. These studies will expand the available information on the structural factors that determine β-lactamase activity in its closely related penicillin hydrolyzing counterparts. The detailed study of these underlying mechanisms is one aspect in understanding how to handle the threatening emergence of multi-resistant bacterial strains and how to target the various β-lactamases that take part in this process. This research is a continuation the work conducted in our laboratory where the structure of the wild-type DAP has been solved [1].

For both mutants crystallization conditions have been found and optimized. Crystals of both DAP mutants diffracted up to 2.4 Å and three datasets were collected at the EMBL-Hamburg X31 and BW7A beam lines (Table 1).

Table 1 Crystallographic parameters and data-collection statistics

Using the wild-type dimer DAP structure as a starting model the structure of DAP 475G487 N275R was solved via molecular replacement. This revealed the presence of 8 monomers in the asymmetric unit cell, which corresponds to a solvent content of 54.10 % and a Matthews coefficient of 2.7 Å3/Da assuming a molecular weight of 56236 Da for one monomer. The refinement of the DAP 475G487 N275R structure is under way and in the near future the structure of DAP 475G487 will also be solved. All crystals used for data-collection were co-crystallized with penicillin G or 6-aminopenicillinic acid. Inspection of the DAP475G487 N275R electron density maps however showed no electron density of the inhibitor. Therefore soaking-experiments of the crystals with these two β-lactam compounds will be conducted in order to obtain the complexes of both mutants with these inhibitors.

References

[1] Y. Asano, Y. Kato, A. Yamada, and C. K. Kondo, Biochemistry 31, 2316 (1992)) [2] C. Bompard-Gilles, H. Remaut, V. Villeret, T. Prangé, L. Fanuel, M. Delmarcelle, B. Joris, J.-M.

Frère, and J. Van Beeumen, Structure 8, 971 (2000) [3] J. R. Knox, P. C. Moews, and J. M. Frère, Chem. Biol. 220, 937 (1996)

DAP 475G487 (1) DAP 475G487(2) DAP 475G487 N275R

Space group P21 P21 P21

a, b, c (Å) and (°)

a= 108.31 b=132.72 c=168.82

=92.00

a=108.14 b=132.44 c=168.53

=91.93

a=109.08 b=132.95 c=169.14

=91.80 Resolution (Å) 30.0-3.0 30.0-2.9 30.0-2.4 No. of unique

reflections 80894 93844 167772

Rsym (%) 11.1 10.7 6.1 Completeness (%) 85.0 89.4 88.5 Redundancy (%) 2.2 2.6 3.0

I/ 7.3 10.1 16.7