THE STRUCTURE OF THE L-AMINOPEPTIDASE/D-AMIDASE/D-ESTERASE FROM OCHROBACTRUM ANTHROPI REVEALS A NEW VARIANT AMONG THE NTN HYDROLASE FOLD

C. Bompard-Gilles¹, V. Villeret¹, L. Fanuel², G.J. Davies³, B. Joris², J.M. Frere² & J. Van Beeumen¹

¹Laboratorium voor Eiwitbiochemie en Eiwitengineering, Rijksuniversiteit-Gent, K.L.Ledeganckstraat, 35, B-9000 Gent, Belgium.
²Laboratoire d'Enzymologie et Centre d'ingénierie des protéines, Université de Liege, Institut de Chimie, B6, B-4000 Sart-Tilman, Belgium.
³Department of Chemistry, University of York, Heslington, York Y01 5DD, UK

Aminopeptidases (alpha aminoacyl-peptide hydrolase) are exopeptidases that catalyse the hydrolysis of the amino-terminal residue from polypeptide substrates. They are mostly divalent cation-dependent or thiol enzymes. Most of them recognize L-amino acid and are of critical biological and medical importance because of their key role in protein modification and degradation. Recently, three aminopeptidases active on peptides containing N-terminal D-residues have been isolated from the bacterium Ochrobactrum anthropi ^1,2. One of them, DmpA, is a L-aminopeptidase showing D-amidasic and D-esterasic activities on peptides containing a N-terminal D-Alanine residue has been cloned and expressed in Escherichia coli². DmpA gene has been sequenced and the deduced amino-acid sequence does not exhibit significant isology with known aminopeptidases². DmpA is synthesized as a single polypeptide precursor but the active form consists of two different peptides resulting from the unique bond cleavage of the precursor polypeptide between Gly249 and Ser250. Site directed mutagenesis studies reveal that residues involved in this bond are essential for protein maturation and catalysis². The cleavage site is recognized in both O. anthropi an E. coli and is similar to that found in enzymes of the N-terminal nucleophile (Ntn) hydrolases superfamily. Their functions, modes of activation and fold have been described previously^3,4,5. These enzymes are amidohydrolases and all are characterized by their unusual use of an N-terminal nucleophile (threonine, serine or cysteine) where its own a-amino group acts as a general base in its catalytic mechanism . This catalytic N-terminal residue is produced by a self catalyzed protein splicing⁵ and is situated at the end of a b-strand. They share a common fold consisting in a core of two stacked antiparallel b-sheets flanked on both sides by helices which provides both the capacity for nucleophilic attack and the possibility of autocatalytic processing³. Fanuel² suggested that DmpA could belong to this family.

We crystallized DmpA using the vapor diffusion method. The crystals belong to the space group P2₁2₁2, with cell parameters a=156.97A, b=96.22A and c=154.41A, diffract to 1.8 A resolution and the asymmetric unit contains six molecules. Multiple isomorphous replacement method was used to determine the structure. This enzyme has a abba fold and is organized in a tetramer in which four equivalent catalytic cavities are formed by the association of three monomers. The structure of DmpA fits well into the Ntn hydrolase pattern and its fold contains most of the helices and strands of the Ntn fold. However, the connectivity of secondary structure elements and b-sheets composition present striking differences with the consensus fold in particular the presence of parallel strands in both sheets.

¹Asano, Y., Nakazawa, A. Kato, Y. & Kondo, K. (1989) J. Biol. Chem. 264: 14233-14239.
²Fanuel, L. PhD Thesis (1997) Université de Liege Belgium.
³Brannigan, J.A., Dodson, G., Duggleby, H.J., Moody, P.C.E., Smith., J.L., Tomchick, D.R. & Murzin, A.G. (1995) Nature 378: 416-419.
⁴Artymuiuk, P.J. (1995) Nature Structural Biology 2: 1035-1037.
⁵Shao, Y. & Kent, S.B.H. (1997) Chemistry and Biology 4: 187-194.