PDBsum entry 3hgx

Lyase

PDB id: 3hgx

Contents

Protein chains

99 a.a. *

Ligands

SAL ×2

PYR ×2

Waters ×16

* Residue conservation analysis

PDB id:

3hgx

Name:

Lyase

Title:

Crystal structure of pseudomonas aeruginosa isochorismate-pyruvate lyase k42a mutant in complex with salicylate and pyruvate

Structure:

Salicylate biosynthesis protein pchb. Chain: a, b. Engineered: yes. Mutation: yes

Source:

Pseudomonas aeruginosa. Organism_taxid: 287. Strain: pao1. Gene: pchb, pa4230. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.50Å R-factor: 0.218 R-free: 0.276

Authors:

Q.Luo,A.L.Lamb

Key ref:

Q.Luo et al. (2009). Structure-function analyses of isochorismate-pyruvate lyase from Pseudomonas aeruginosa suggest differing catalytic mechanisms for the two pericyclic reactions of this bifunctional enzyme. Biochemistry, 48, 5239-5245. PubMed id: 19432488

Date:

14-May-09 Release date: 30-Jun-09

Protein chains

Q51507  (PCHB_PSEAE) -  Isochorismate pyruvate lyase from Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)

Seq: 101 a.a.

Struc: 99 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 2: E.C.4.2.99.21 - isochorismate lyase.
• Reaction: isochorismate = salicylate + pyruvate

isochorismate = salicylate (Bound ligand (Het Group name = PYR) corresponds exactly) + pyruvate (Bound ligand (Het Group name = SAL) corresponds exactly)

• Enzyme class 3: E.C.5.4.99.5 - chorismate mutase.

Pathway:

• Reaction: chorismate = prephenate

chorismate (Bound ligand (Het Group name = SAL) matches with 52.94% similarity) = prephenate

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

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

reference

Biochemistry 48:5239-5245 (2009)

PubMed id: 19432488

Structure-function analyses of isochorismate-pyruvate lyase from Pseudomonas aeruginosa suggest differing catalytic mechanisms for the two pericyclic reactions of this bifunctional enzyme.

Q.Luo, J.Olucha, A.L.Lamb.

ABSTRACT

The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) catalyzes two pericyclic reactions in a single active site. PchB physiologically produces salicylate and pyruvate from isochorismate for ultimate incorporation of the salicylate into the siderophore pyochelin. PchB also produces prephenate from chorismate, most likely due to structural homology to the Escherchia coli chorismate mutase. The molecular basis of catalysis among enzymatic pericyclic reactions is a matter of debate, one view holding that catalysis may be derived from electrostatic transition state stabilization and the opposing view that catalysis is derived from the generation of a reactive substrate conformation. Mutant forms of PchB were generated by site-directed mutagenesis at the site (K42) hypothesized to be key for electrostatic transition state stabilization (K42A, K42Q, K42E, and K42H). The loop containing K42 is mobile, and a mutant to slow loop dynamics was also designed (A43P). Finally, a previously characterized mutation (I87T) was also produced. Circular dichroism was used to assess the overall effect on secondary structure as a result of the mutations, and X-ray crystallographic structures are reported for K42A with salicylate and pyruvate bound and for apo-I87T. The data illustrate that the active site architecture is maintained in K42A-PchB, which indicates that differences in activity are not caused by secondary structural changes or by differences in active site loop conformation but rather by the chemical nature of this key residue. In contrast, the I87T structure demonstrates considerable mobility, suggesting that loop dynamics and conformational plasticity may be important for efficient catalysis. Finally, the mutational effects on k(cat) provide evidence that the two activities of PchB are not covariant and that a single hypothesis may not provide a sufficient explanation for catalysis.