Article

2-Hydroxychromene-2-carboxylic acid isomerase: a kappa class glutathione transferase from Pseudomonas putida.

Department of Biochemistry, Center in Molecular Toxicology, and Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Biochemistry (impact factor: 3.42). 07/2007; 46(23):6710-22. DOI:10.1021/bi700356u
Source: PubMed

ABSTRACT The enzyme 2-hydroxychromene-2-carboxylic acid (HCCA) isomerase catalyzes the glutathione (GSH)-dependent interconversion (Keq = 1.5) of HCCA and trans-o-hydroxybenzylidene pyruvic acid (tHBPA) in the naphthalene catabolic pathway of Pseudomonas putida. The dimeric protein binds one molecule of GSH very tightly (Kd approximately 5 nM) and a second molecule of GSH with much lower affinity (Kd approximately 2 to 11 microM). The enzyme is unstable in the absence of GSH. The turnover number in the forward direction (47 s(-1) at 25 degrees C) greatly exceeds off rates for GSH (koff approximately 10(-3) to 10(-2) s(-1) at 10 degrees C), suggesting that GSH acts as a tightly bound cofactor in the reaction. The crystal structure of the enzyme at 1.7 A resolution reveals that the isomerase is closely related to class kappa GSH transferases. Diffraction quality crystals could only be obtained in the presence of GSH and HCCA/tHBPA. Clear electron density is seen for GSH. Electron density for the organic substrates is located near the GSH and is best modeled to include both HCCA and tHBPA at occupancies of 0.5 for each. Although there is no electron density connecting the sulfur of GSH to the organic substrates, the sulfur is located very close (2.78 A) to C7 of HCCA. Taken together, the results suggest that the isomerization reaction involves a short-lived covalent adduct between the sulfur of GSH and C7 of the substrate.

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Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

10 degrees C
 
11 microM
 
25 degrees C
 
bound cofactor
 
class kappa GSH transferases
 
Clear electron density
 
dimeric protein binds
 
enzyme 2-hydroxychromene-2-carboxylic acid
 
GSH
 
GSH acts
 
GSH)-dependent interconversion
 
isomerization reaction
 
lower affinity
 
naphthalene catabolic pathway
 
organic substrates
 
Pseudomonas putida
 
second molecule
 
substrate
 
trans-o-hydroxybenzylidene pyruvic acid
 
turnover number