Glutaryl-7-aminocephalosporanic-acid acylase

 

Glutaryl 7-aminocephalosporanic acid (7-ACA) acylase catalyses the hydrolysis of Glutaryl 7-ACA to Glutaric acid and 7-ACA. It belongs to the family of N-terminal hydrolase (peptidase S45 family). 7-ACA is the starting compound for the synthesis of cephalosporin antibiotics and it is usually obtained industrially by chemical deacylation of cephalosporin C(CPC). Since the chemical production of 7-ACA is expensive and produce toxic chemical wastes, the enzymatic conversion of CPC to 7-ACA by 7-ACA acylase is of great interest.

In order for the enzyme to be activated towards catalysing the hydrolysis of the glutaryl form of 7-aminocephalosporanic acid, autoproteolysis must occur at the nucleophilic Ser170, revealing an N terminal nucleophile capable of inducing nucleophilic attack at the substrate's glutaryl carbonyl. The process forms two descrete proteins chains. The crystal structure depicts the non-cleaved, precursor protein, whereas the active protein froms a heterotetramer from the two resulting chains.

 

Reference Protein and Structure

Sequence
P07662 UniProt (3.5.1.93) IPR014395 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas sp. SY-77-1 (Bacteria) Uniprot
PDB
3s8r - Crystal Structures of Glutaryl 7-Aminocephalosporanic Acid Acylase: Insight into Autoproteolytic Activation (2.5 Å) PDBe PDBsum 3s8r
Catalytic CATH Domains
3.60.20.10 CATHdb (see all for 3s8r)
Cofactors
Water (1)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.93)

(7R)-7-(4-carboxylatobutanamido)cephalosporanate
CHEBI:58693ChEBI
+
water
CHEBI:15377ChEBI
→
7beta-aminocephalosporanic acid zwitterion
CHEBI:58501ChEBI
+
glutarate(2-)
CHEBI:30921ChEBI
Alternative enzyme names: 7-beta-(4-carboxybutanamido)cephalosporanic acid acylase, Cephalosporin C acylase, Glutaryl-7-ACA acylase, CA, GCA, GA, Cephalosporin acylase, Glutaryl-7-aminocephalosporanic acid acylase, GL-7-ACA acylase,

Enzyme Mechanism

Introduction

Ser170 alpha-amino group acts as a base to deprotonate its own hydroxyl group, which nucleophilically attacks the carbonyl group of the substrate. This results in the formation of a covalent enzyme-substrate transition state, stabilised by the oxyanion hole formed by the mainchain nitrogen atom of Val239 and the side chain of Asn413. Alpha-amino group of Ser170 then protonates the leaving group and deprotonates a water molecule to allow it to restore the enzyme by a nucleophilic attack to the acylenzyme.

Catalytic Residues Roles

UniProt PDB* (3s8r)
Ser199 (N-term) Ala170A (N-term) Its alpha-amino group acts as a base to deprotonate its own. The alpha-amino group of Ser 170 then protonates the leaving group and deprotonates a water molecule to allow it to restore the enzyme by a nucleophilic attack to the acylenzyme. proton acceptor, steric role, proton donor
His221, Glu653 His192A, Glu624A The interactions between His192 and Glu624 help orientate the nucleophilic Ser170 towards the substrate's glutaryl group. The protonation state of the N terminus is also mediated through hydrogen bonding interactions between the amine nitrogen, His192A and Glu624A which work in partnership as a catalytic dyad. This dyad is similar to that seen for serine proteases, although the hydrogen bond to Ser170A involves the terminal amine group, rather than the side chain hydroxy. activator, increase acidity, steric role, electrostatic stabiliser
Val268 (main-N), Asn442 Val239A (main-N), Asn413A Forms the oxyanion hole to stabilise the transition state. electrostatic stabiliser
Ser199 Ala170A Acts as a catalytic nucleophile. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleofuge, polar interaction, proton donor, nucleophile, proton acceptor, activator, increase electrophilicity
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, proton relay, bimolecular nucleophilic addition, enzyme-substrate complex formation, intramolecular elimination, intermediate formation, overall product formed, unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, native state of enzyme regenerated, inferred reaction step

References

  1. Kim JK et al. (2003), Biochemistry, 42, 4084-4093. Crystal Structures of Glutaryl 7-Aminocephalosporanic Acid Acylase:  Insight into Autoproteolytic Activation†. DOI:10.1021/bi027181x. PMID:12680762.
  2. Pollegioni L et al. (2013), Appl Microbiol Biotechnol, 97, 2341-2355. Cephalosporin C acylase: dream and(/or) reality. DOI:10.1007/s00253-013-4741-0. PMID:23417342.
  3. Fritz-Wolf K et al. (2002), Protein Sci, 11, 92-103. Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C. DOI:10.1110/ps.27502. PMID:11742126.
  4. Kim Y et al. (2001), Chem Biol, 8, 1253-1264. Structure of cephalosporin acylase in complex with glutaryl-7-aminocephalosporanic acid and glutarate: insight into the basis of its substrate specificity. DOI:10.1016/s1074-5521(01)00092-8. PMID:11755403.
  5. Kim Y et al. (2000), Structure, 8, 1059-1068. The 2.0 Å Crystal Structure of Cephalosporin Acylase. DOI:10.1016/s0969-2126(00)00505-0. PMID:11080627.

Step 1. A water molecule acts as a proton carrier in the deprotonation of the Ser170A side chain hydroxyl by the N terminal amine group. The presence of low barrier hydrogen bonds between Ser170, His193, Glu525 and the conserved water molecule facilitates proton transfer by lowering the activation energy associated with each step.

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Catalytic Residues Roles

Residue Roles
Ala170A hydrogen bond donor
His192A steric role
Glu624A steric role
His192A electrostatic stabiliser
Glu624A electrostatic stabiliser
His192A activator
Glu624A activator
Ala170A proton donor
Ala170A (N-term) proton acceptor

Chemical Components

proton transfer, proton relay

Step 2. The side chain hydroxide of Ser170A attacks the substrate amide carbonyl, forming a tetrahedral intermediate.

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Catalytic Residues Roles

Residue Roles
Ala170A hydrogen bond acceptor, polar interaction, hydrogen bond donor
Ala170A (N-term) steric role
His192A steric role
Glu624A steric role
Val239A (main-N) electrostatic stabiliser
Asn413A electrostatic stabiliser
Ala170A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation

Step 3. The tetrahedral intermediate collapses, forming a serine linked glutarate intermediate and eliminating 7-Aminocephalosporanic acid.

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Catalytic Residues Roles

Residue Roles
Ala170A covalently attached, hydrogen bond donor, increase electrophilicity
His192A electrostatic stabiliser
Glu624A electrostatic stabiliser
Val239A (main-N) electrostatic stabiliser
Asn413A electrostatic stabiliser
His192A activator
Glu624A activator
Ala170A (N-term) proton donor

Chemical Components

ingold: intramolecular elimination, proton transfer, intermediate formation, overall product formed

Step 4. Water is activated by the non-protonated terminal amine of Ser170 to attack at the enzyme-substrate intermediate's carbonyl group, forming a second tetrahedral intermediate.

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Catalytic Residues Roles

Residue Roles
Ala170A covalently attached
Val239A (main-N) electrostatic stabiliser
Asn413A electrostatic stabiliser
His192A activator
Glu624A activator
Ala170A increase electrophilicity
His192A electrostatic stabiliser
Glu624A electrostatic stabiliser
Ala170A (N-term) proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation, overall product formed

Step 5. The tetrahedral anion intermediate collapses, releasing glutarate and the N terminal serine.

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Catalytic Residues Roles

Residue Roles
Ala170A covalently attached, activator, hydrogen bond donor
His192A electrostatic stabiliser
Val239A (main-N) electrostatic stabiliser
Asn413A electrostatic stabiliser
Glu624A electrostatic stabiliser
Ala170A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, enzyme-substrate complex cleavage

Step 6. The anionic serine side chain undergoes an intamolecular deprotonation, removing a proton from the terminal amine group via a conserved water molecule.

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Catalytic Residues Roles

Residue Roles
Ala170A hydrogen bond acceptor, hydrogen bond donor
His192A electrostatic stabiliser
Glu624A electrostatic stabiliser
His192A increase acidity
Glu624A increase acidity
Ala170A proton acceptor
Ala170A (N-term) proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step, proton relay
Download: Image, Marvin File

Step 1. A water molecule acts as a proton carrier in the deprotonation of the Ser170A side chain hydroxyl by the N terminal amine group. The presence of low barrier hydrogen bonds between Ser170, His193, Glu525 and the conserved water molecule facilitates proton transfer by lowering the activation energy associated with each step.

Step 2. The side chain hydroxide of Ser170A attacks the substrate amide carbonyl, forming a tetrahedral intermediate.

Step 3. The tetrahedral intermediate collapses, forming a serine linked glutarate intermediate and eliminating 7-Aminocephalosporanic acid.

Step 4. Water is activated by the non-protonated terminal amine of Ser170 to attack at the enzyme-substrate intermediate's carbonyl group, forming a second tetrahedral intermediate.

Step 5. The tetrahedral anion intermediate collapses, releasing glutarate and the N terminal serine.

Step 6. The anionic serine side chain undergoes an intamolecular deprotonation, removing a proton from the terminal amine group via a conserved water molecule.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Mei Leung, Charity Hornby