Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification.
GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC I's active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.
Prabhakar
Vikas
V
Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Capila
Ishan
I
Bosques
Carlos J
CJ
Pojasek
Kevin
K
Sasisekharan
Ram
R
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D001665:Binding Sites; D019765:Chondroitin ABC Lyase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular; D006025:Glycosaminoglycans / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011514:Proteus vulgaris / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D006025:Glycosaminoglycans; D011993:Recombinant Fusion Proteins; D019765:Chondroitin ABC Lyase