Fusarinine-C ornithinesterase
EstB is an esterase from Burkholderia gladioli whose physiological function is the hydrolysis of an N5-acyl-L-ornithine ester with moderate S-enantioselectivity.
It is also known to act on short-chain (C4-C6) fatty acid esters and triglycerides, including tertiary alcohol esters (e.g. linalyl acetate). It also shows activity on p-nitrophenyl esters is generally higher than on o-nitrophenyl esters. The Burkholderia gladioli protein is known to be strongly inhibited by eserin, NaF, HgCl2, SDS and Triton X-100.
The primary structure exhibits homology to esterases from family VIII, which are related to class C beta-lactamases. However, catalytic activity is located within the 'beta-lactamase' motif and not the 'esterase' motif. Despite structural similarity with peptidases and class C beta-lactamases, EstB shows no activity with peptides or beta-lactamases.
Reference Protein and Structure
- Sequence
-
Q9KX40
(3.1.1.-)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Burkholderia gladioli (Bacteria)
- PDB
-
1ci8
- ESTERASE ESTB FROM BURKHOLDERIA GLADIOLI: AN ESTERASE WITH (BETA)-LACTAMASE FOLD.
(2.0 Å)
- Catalytic CATH Domains
-
3.40.710.10
(see all for 1ci8)
Enzyme Reaction (EC:3.1.1.48)
+
→
+
+
Alternative enzyme names: Ornithine esterase, 5-N-acyl-L-ornithine-ester hydrolase,
Enzyme Mechanism
Introduction
Computational studies on the pKa and proton relay chains in the active site of EstB have suggested the following mechanism:
- Ser75 is activated by bonds to Tyr181 (held in place by H-bonds with Lys 78 and Trp 348 side chains). Ser75 is deprotonated by a proton relay chain that involves Lys78, Tyr133, HOH and Asp186. Once activated, Ser75 initiates the nucleophilic attack on the substrate. The main chain amide groups of Ser 75 and Val 351 can hydrogen bond to the ester substrate, activating the ester bond as an electrophile.
- The intermediate collapses, acylating Ser 75. The hydroxyl leaving group is protonated through the same proton relay chain as in step 1.
- Deacylation of the acyl-enzyme is by hydrolysis, the catalytic residues playing the same roles as they did in the acylation steps.
Catalytic Residues Roles
| UniProt | PDB* (1ci8) | ||
| Ser75 | Ser75A | The Ser 75 sidechain is the nucleophile, attacking the substrate ester, cleaving the substrate and acylating itself. | covalent catalysis, proton shuttle (general acid/base) |
| Lys78, Tyr181 | Lys78A, Tyr181A | Helps activate Ser75. | activator, proton shuttle (general acid/base) |
| Val351 (main-N), Ser75 (main-N) | Val351A (main-N), Ser75A (main-N) | The main chain amide hydrogen bonds to the ester substrate, and acyl-enzyme intermediate, activating them as electrophiles and acting as an oxyanion hole during the transition states and intermediates. | electrostatic stabiliser |
| Trp348 | Trp348A | Helps to appropriately position Tyr181. | steric role |
| His253, Val351, Leu135, Ile152 | His253A, Val351A, Leu135A, Ile152A | Thought to be critical for the S enantioselectivity of the protein. | steric role |
| Asp186, Tyr133 | Asp186A, Tyr133A | Forms part of the proton relay chain that deprotonates the nucleophilic Ser75 residue. The chain is: Lys78-Tyr133-HOH-Asp186. | proton shuttle (general acid/base) |
*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
References
- Chen L et al. (2011), J Phys Chem B, 115, 13019-13025. Theoretical Study of the Mechanism of Proton Transfer in the Esterase Estb fromBurkholderia Gladioli. DOI:10.1021/jp206297d. PMID:21910435.
- Ivancic M et al. (2007), J Biotechnol, 129, 109-122. Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution. DOI:10.1016/j.jbiotec.2006.10.007. PMID:17147964.
- Wagner UG et al. (2002), Protein Sci, 11, 467-478. EstB from Burkholderia gladioli: A novel esterase with a β-lactamase fold reveals steric factors to discriminate between esterolytic and β-lactam cleaving activity. DOI:10.1110/ps.33002. PMID:11847270.
- Petersen EI et al. (2001), J Biotechnol, 89, 11-25. A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to β-lactamases and dd-peptidases. DOI:10.1016/s0168-1656(01)00284-x. PMID:11472796.
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Ser75A | covalent catalysis, proton shuttle (general acid/base) |
| Asp186A | proton shuttle (general acid/base) |
| Lys78A | proton shuttle (general acid/base) |
| Tyr133A | proton shuttle (general acid/base) |
| Ser75A (main-N) | electrostatic stabiliser |
| Val351A (main-N) | electrostatic stabiliser |
| Tyr181A | activator |
| Lys78A | activator |
| Trp348A | steric role |
| Leu135A | steric role |
| Val351A | steric role |
| His253A | steric role |
| Ile152A | steric role |
Chemical Components
Introduction
Despite the lack of beta-lactamase activity, the mechanism of EstB is thought to be similar to that of the similar P99 beta-lactamase. The mechanism can be described as follows:
- Lys 78 and Trp 348 side chains hydrogen bond to Tyr 181, decreasing Tyr 181 pKa and allowing Tyr 181 to exist as the phenolate anion. Tyr 181 can then activate Ser 75 as a nucleophile by deprotonation.
- The main chain NH groups of Ser 75 and Val 351 can hydrogen bond to the ester substrate, activating the ester bond as an electrophile.
- Ser 75 attacks the carbon of the substrate ester. A tetrahedral intermediate is formed. Ser 75 and Val 351 main chain NHs stabilise the charge on this intermediate and the preceding transition state.
- The intermediate collapses, acylating Ser 75. The hydroxyl leaving group is protonated by Tyr 181.
- Deacylation of the acyl-enzyme is by hydrolysis, the catalytic residues playing the same roles as they did in the acylation steps.
Catalytic Residues Roles
| UniProt | PDB* (1ci8) | ||
| Ser75 | Ser75A | The Ser 75 sidechain is the nucleophile, attacking the substrate ester, cleaving the substrate and acylating itself. | covalent catalysis, proton shuttle (general acid/base) |
| Tyr181 | Tyr181A | Tyr 181, as the phenolate anion, is the base that deprotonates Ser 75 for better nucleophilic attack; the proton is transferred to the substrate leaving group. In the deacylation reaction Tyr 181 deprotonates water and transfers the proton to Ser 75. | proton shuttle (general acid/base) |
| Val351 (main-N), Ser75 (main-N) | Val351A (main-N), Ser75A (main-N) | The main chain amide hydrogen bonds to the ester substrate, and acyl-enzyme intermediate, activating them as electrophiles and acting as an oxyanion hole during the transition states and intermediates. | electrostatic stabiliser |
| Lys78, Trp348 | Lys78A, Trp348A | Proximal to the general acid/base tyrosine (Tyr181), decreasing its pKa. | activator, electrostatic stabiliser |
| His253, Val351, Leu135, Ile152 | His253A, Val351A, Leu135A, Ile152A | Thought to be critical for the S enantioselectivity of the protein. | steric role |
| Asp186, Tyr133 | Asp186A, Tyr133A | Not thought to be active in this mechanism proposal. | hydrogen bond acceptor |
*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
References
- Wagner UG et al. (2002), Protein Sci, 11, 467-478. EstB from Burkholderia gladioli: A novel esterase with a β-lactamase fold reveals steric factors to discriminate between esterolytic and β-lactam cleaving activity. DOI:10.1110/ps.33002. PMID:11847270.
- Ivancic M et al. (2007), J Biotechnol, 129, 109-122. Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution. DOI:10.1016/j.jbiotec.2006.10.007. PMID:17147964.
- Petersen EI et al. (2001), J Biotechnol, 89, 11-25. A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to β-lactamases and dd-peptidases. DOI:10.1016/s0168-1656(01)00284-x. PMID:11472796.
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Leu135A | steric role |
| Ile152A | steric role |
| His253A | steric role |
| Val351A | steric role |
| Tyr181A | proton shuttle (general acid/base) |
| Ser75A | covalent catalysis |
| Ser75A (main-N) | electrostatic stabiliser |
| Ser75A | proton shuttle (general acid/base) |
| Val351A | electrostatic stabiliser |
| Lys78A | electrostatic stabiliser |
| Trp348A | activator, electrostatic stabiliser |
| Lys78A | activator |
| Val351A (main-N) | electrostatic stabiliser |
| Tyr133A | hydrogen bond acceptor, hydrogen bond donor |
| Asp186A | hydrogen bond acceptor |