Database accession: MF7000163
Name: Isochorismate-pyruvate lyase, K42A mutant (Pseudomonas aeruginosa)
PDB ID: 3hgx
Experimental method: X-ray (2.50 Å)
Assembly: Homodimer
Source organism: Pseudomonas aeruginosa
Primary publication of the structure:
Luo Q, Olucha J, Lamb AL
Structure-function analyses of isochorismate-pyruvate lyase from Pseudomonas aeruginosa suggest differing catalytic mechanisms for the two pericyclic reactions of this bifunctional enzyme.
(2009) Biochemistry 48: 5239-45
PMID: 19432488
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.
Annotations from the GeneOntology database. Only terms that fit at least two of the interacting proteins are shown. Molecular function:
carbon-oxygen lyase activity carbon-oxygen lyase activity
chorismate mutase activity chorismate mutase activity
isochorismate pyruvate lyase activity isochorismate pyruvate lyase activity
Biological process:
chorismate metabolic process chorismate metabolic process
pyochelin biosynthetic process pyochelin biosynthetic process
salicylic acid biosynthetic process salicylic acid biosynthetic process
Cellular component: not assigned
Structural annotations of the participating protein chains.Entry contents: 2 distinct polypeptide molecules
Chains: A, B
Notes: All chains according to the most probable oligomerization state stored in PDBe were considered.
Number of unique protein segments: 1
Name: Isochorismate pyruvate lyase
Source organism: Pseudomonas aeruginosa
Length: 101 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMKTPEDCTGLADIREAIDRIDLDIVQALGRRMDYVKAASRFKASEAAIPAPERVAAMLPERARWAEENGLDAPFVEGLFAQIIHWYIAEQIKYWRQTRGAA
UniProtKB AC: Q51507 (positions: 1-99)
Coverage: 98%
Name: Isochorismate pyruvate lyase
Source organism: Pseudomonas aeruginosa
Length: 101 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMKTPEDCTGLADIREAIDRIDLDIVQALGRRMDYVKAASRFKASEAAIPAPERVAAMLPERARWAEENGLDAPFVEGLFAQIIHWYIAEQIKYWRQTRGAA
UniProtKB AC: Q51507 (positions: 1-99)
Coverage: 98%
Evidence demonstrating that the participating proteins are unstructured prior to the interaction and their folding is coupled to binding. Representative domain in related structures: Chorismate mutase type II
Evidence level: Indirect evidence
Evidence coverage: The full structure participates in mutual synergistic folding.
Complex Evidence:
The enzyme is an intertwined dimer of three helices with connecting loops. The N-terminal helices of the two monomers twine together to form an anti-parallel coiled-coil with a hydrophobic interaction surface. The loop between the first and second helices is disordered (PMID:16914555).
Chain A:
N/A
Chain B:
N/A
Surface and contacts features:
Structures from the PDB that contain the same number of proteins, and the proteins from the two structures show a sufficient degree of pairwise similarity, i.e. they belong to the same UniRef90 cluster (the full proteins exhibit at least 90% sequence identity) and convey roughly the same region to their respective interactions (the two regions from the two proteins share a minimum of 70% overlap). Download the CIF file (.cif)
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