PDBsum entry 2adv

Hydrolase

PDB id: 2adv

Contents

Protein chains

161 a.a. *

28 a.a. *

494 a.a. *

Waters ×374

* Residue conservation analysis

PDB id:

2adv

Name:

Hydrolase

Title:

Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: mutational study of activation mechanism

Structure:

Glutaryl 7- aminocephalosporanic acid acylase. Chain: a. Fragment: alpha domain. Engineered: yes. Glutaryl 7- aminocephalosporanic acid acylase. Chain: b. Fragment: beta1 domain. Engineered: yes. Glutaryl 7- aminocephalosporanic acid acylase.

Source:

Pseudomonas sp.. Organism_taxid: 405038. Strain: gk16. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PQS)

Resolution:

2.24Å R-factor: 0.185 R-free: 0.210

Authors:

J.K.Kim,I.S.Yang,H.J.Shin,K.J.Cho,E.K.Ryu,S.H.Kim,S.S.Park,K.H.Kim

Key ref:

J.K.Kim et al. (2006). Insight into autoproteolytic activation from the structure of cephalosporin acylase: a protein with two proteolytic chemistries. Proc Natl Acad Sci U S A, 103, 1732-1737. PubMed id: 16446446 DOI: 10.1073/pnas.0507862103

Date:

21-Jul-05 Release date: 24-Jan-06

Protein chain

P07662  (G7AC_PSEU7) -  Glutaryl-7-aminocephalosporanic-acid acylase from Pseudomonas sp. (strain SY-77)

Seq: 720 a.a.

Struc: 161 a.a.*

Protein chain

P07662  (G7AC_PSEU7) -  Glutaryl-7-aminocephalosporanic-acid acylase from Pseudomonas sp. (strain SY-77)

Seq: 720 a.a.

Struc: 28 a.a.

Protein chain

P07662  (G7AC_PSEU7) -  Glutaryl-7-aminocephalosporanic-acid acylase from Pseudomonas sp. (strain SY-77)

Seq: 720 a.a.

Struc: 494 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B, C: E.C.3.5.1.93 - glutaryl-7-aminocephalosporanic-acid acylase.
• Reaction: (7R)-7-(4-carboxybutanamido)cephalosporanate + H2O = (7R)-7- aminocephalosporanate + glutarate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1073/pnas.0507862103

Proc Natl Acad Sci U S A 103:1732-1737 (2006)

PubMed id: 16446446

Insight into autoproteolytic activation from the structure of cephalosporin acylase: a protein with two proteolytic chemistries.

J.K.Kim, I.S.Yang, H.J.Shin, K.J.Cho, E.K.Ryu, S.H.Kim, S.S.Park, K.H.Kim.

ABSTRACT

Cephalosporin acylase (CA), a member of the N-terminal nucleophile hydrolase family, is activated through sequential primary and secondary autoproteolytic reactions with the release of a pro segment. We have determined crystal structures of four CA mutants. Two mutants are trapped after the primary cleavage, and the other two undergo secondary cleavage slowly. These structures provide a look at pro-segment conformation during activation in N-terminal nucleophile hydrolases. The highly strained helical pro segment of precursor is transformed into a relaxed loop in the intermediates, suggesting that the relaxation of structural constraints drives the primary cleavage reaction. The secondary autoproteolytic step has been proposed to be intermolecular. However, our analysis provides evidence that CA is processed in two sequential steps of intramolecular autoproteolysis involving two distinct residues in the active site, the first a serine and the second a glutamate.

Selected figure(s)

Figure 2.
Structural comparison of CA proteins. (A) Superposition of the overall structures of S170A, S170C, E159Q, Y202L, R226K, and the mature CA. Each structure is shown in yellow, cyan, dark green, green, slate blue, and marine, respectively. To emphasize the differences of the pro segments, the pro segment of Y202L is shown in red. Residues Gly-160, Ser-170, Tyr-202, and Arg-226 are shown in a ball-and-stick representation in magenta. (B) Stereoview of the pro-segment conformation of Y202L. The electron density map corresponds to the 2 F [o] − F [c] map calculated at 0.5 σ.

Figure 3.
Secondary autocleavage site of CA proteins. The interactions between pro-segment and protein residues in Y202L, precursor S170A, R226K, S170C, and E159Q. Those involved in hydrophobic interactions in S170A are superimposed in a transparent space-filling representation. Hydrogen bonds are indicated by dotted lines, and water molecules are represented as red spheres. The distances between the side chain of Glu-159 and the backbone carbon atom of Gly-160 via WAT1 in Y202L and R226K are highlighted by thick dotted lines.

Figures were selected by the author.

Author's comment

Two chemistry in cephalosporin acylase: the precursor is activated through sequential primary serine proteolysis and secondary acidic proteolysis