3HGX

Crystal Structure of Pseudomonas aeruginosa Isochorismate-Pyruvate Lyase K42A mutant in complex with salicylate and pyruvate

Summary for 3HGX

• Entry DOI: 10.2210/pdb3hgx/pdb
• Related: 2H9C 2H9D 3HGW
• Descriptor: Salicylate biosynthesis protein pchB, 2-HYDROXYBENZOIC ACID, PYRUVIC ACID, ... (4 entities in total)
• Functional Keywords: siderophore biosynthesis, pyochelin, isochorismate-pyruvate lyase, chorismate mutase, pchb, lyase
• Biological source: Pseudomonas aeruginosa
• Total number of polymer chains: 2
• Total formula weight: 23230.29

Authors

Luo, Q.,Lamb, A.L. (deposition date: 2009-05-14, release date: 2009-06-30, Last modification date: 2023-11-15)

Primary citation

Luo, Q.,Olucha, J.,Lamb, A.L.
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, 2009

PubMed 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.

PubMed: 19432488
DOI: 10.1021/bi900456e

Experimental method

X-RAY DIFFRACTION (2.5 Å)