Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation.
Glutaryl 7-aminocephalosporanic acid acylase (GCA, EC 3.5.1.11) is a member of N-terminal nucleophile (Ntn) hydrolases. The native enzyme is an (alpha beta)(2) heterotetramer originated from an enzymatically inactive precursor of a single polypeptide. The activation of precursor GCA consists of primary and secondary autoproteolytic cleavages, generating a terminal residue with both a nucleophile and a base and releasing a nine amino acid spacer peptide. We have determined the crystal structures of the recombinant selenomethionyl native and S170A mutant precursor from Pseudomonas sp. strain GK16. Precursor activation is likely triggered by conformational constraints within the spacer peptide, probably inducing a peptide flip. Autoproteolytic site solvent molecules, which have been trapped in a hydrophobic environment by the spacer peptide, may play a role as a general base for nucleophilic attack. The activation results in building up a catalytic triad composed of Ser170/His192/Glu624. However, the triad is not linked to the usual hydroxyl but the free alpha-amino group of the N-terminal serine residue of the native GCA. Mutagenesis and structural data support the notion that the stabilization of a transient hydroxazolidine ring during autoproteolysis would be critical during the N --> O acyl shift. The autoproteolytic activation mechanism for GCA is described.
Kim
Jin Kwang
JK
Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea.
Yang
In Seok
IS
Rhee
Sangkee
S
Dauter
Zbigniew
Z
Lee
Young Sik
YS
Park
Sung Soo
SS
Kim
Kyung Hyun
KH
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase