CRYSTAL STRUCTURE OF CARBOXYPEPTIDASE G2 AND COMPARISON WITH OTHER ZINC-CONTAINING EXOPEPTIDASES. Siân Rowsell1, Richard A. Pauptit2, Alec D. Tucker2, Peter Brick1, Lesley F. Lloyd1, Roger G. Melton3, David M. Blow1. 1Blackett Laboratory, Imperial College, London SW7 2BZ, UK, 2Protein Structure Laboratory, Zeneca Pharmaceuticals, Macclesfield, Cheshire, SK10 4TG, UK, 3Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, SP4 0JG, UK
Enzymes of the carboxypeptidase G class hydrolyse the C-terminal glutamate moiety from folic acid and analogues such as methotrexate. Carboxypeptidase G2 (CPG2) is a zinc metalloenzyme produced by Pseudomonas sp. strain RS-16. There is no amino-acid sequence homology with other carboxypeptidases for which structural information is available.
Cancer therapies often rely on the plasma depletion of reduced folates which are essential cofactors in purine and pyrimidine biosynthesis. When coupled to antibodies which target the tumour cells, CPG2 is potentially useful in activating prodrugs at the tumour site. CPG2 may also be used to remove excess methotrexate from circulation in patients having prolonged treatments with this relatively toxic agent.
The crystal structure of CPG2 has been determined to 3.0 Å resolution by the technique of multiple isomorphous replacement. The current crystallographic R-factor is 21.9% for all reflections between 10.0 and 3.0 Å resolution.
The CPG2 molecule is a dimer composed of subunits of molecular mass 41,800 Da. The subunit consists of a catalytic domain, which contains a cocatalytic zinc site, and a second domain, which forms a dimer interface through hydrophobic interactions as well as through hydrogen bonding between two symmetry-related [[beta]]-strands. The catalytic domain has close structural similarity to several zinc-containing exopeptidases. The topology of the second domain is similar to several RNA-binding domains. A structural comparison between CPG2 and other zinc-containing exopeptidases suggests a remote divergent evolutionary relationship between these enzymes.