Structure of an aryl esterase from Pseudomonas fluorescens
Jeremy D Cheeseman1, Ante Tocilj, Seongsoon Park
1Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreál, Québec H3A 2K6, Canada.
Summary
The structure of Pseudomonas fluorescens esterase (PFE) was determined using X-ray diffraction. This aryl esterase exhibits structural similarities to bacterial haloperoxidases and possesses a unique, occluded active site.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Pseudomonas fluorescens esterase (PFE) is an enzyme with known esterase activity.
- PFE also exhibits secondary enzymatic functions, including low bromoperoxidase activity.
- Understanding the structural basis of PFE's activity is crucial for enzyme engineering and functional studies.
Purpose of the Study:
- To determine the high-resolution three-dimensional structure of PFE.
- To elucidate the structural basis for PFE's esterase and bromoperoxidase activities.
- To compare the structure of PFE with related enzymes, particularly haloperoxidases and other esterases.
Main Methods:
- X-ray diffraction crystallography was employed to solve the structure of PFE.
- The resolution achieved for the PFE structure was 1.8 Å.
- Structural comparisons were performed using root-mean-square deviation (r.m.s.d.) calculations for Cα atoms.
Main Results:
- The crystal structure of PFE reveals a characteristic alpha/beta-hydrolase fold.
- PFE displays significant structural similarity to non-heme bacterial haloperoxidases (average r.m.s.d. of 0.8 Å over 271 Cα atoms).
- The active site of PFE is notably occluded, with specific residues (Trp28, Met95) restricting the acyl-binding pocket, explaining its preference for small acyl groups.
Conclusions:
- PFE possesses an alpha/beta-hydrolase fold and shares structural features with bacterial haloperoxidases.
- The occluded active site and specific residues dictate PFE's substrate specificity for small acyl groups.
- The structural data provides insights into the dual functionality and substrate preference of PFE.
Related Concept Videos
02:43E1 Reaction: Stereochemistry and Regiochemistry
One of the critical aspects of the E1 reaction mechanism, as also observed in E2, is the regiochemistry, with multiple regioisomers obtained as products. In the example discussed, the presence of water as a weak base favors elimination over substitution to generate two alkenes. Given that alkenes’ stability increases with the number of alkyl groups across the double bond, typically, E1 reactions lead to the Zaitsev product, for this is more substituted and stable than the Hofmann product.
01:12Acid Halides to Esters: Alcoholysis
Alcoholysis is a nucleophilic acyl substitution reaction in which an alcohol functions as a nucleophile. Acid halides react with alcohol to produce esters. The mechanism proceeds in three steps:
01:13Esters to Carboxylic Acids: Acid-Catalyzed Hydrolysis
Hydrolysis of esters under acidic conditions proceeds through a nucleophilic acyl substitution. In the presence of excess water, the reaction proceeds in a reversible manner, forming carboxylic acids and alcohols.
During hydrolysis, the ester is first activated towards nucleophilic attack through the protonation of the carboxyl oxygen atom by the acid catalyst. The protonation makes the ester carbonyl carbon more electrophilic. In the next step, water acts as a nucleophile and adds to the...
During hydrolysis, the ester is first activated towards nucleophilic attack through the protonation of the carboxyl oxygen atom by the acid catalyst. The protonation makes the ester carbonyl carbon more electrophilic. In the next step, water acts as a nucleophile and adds to the...
01:30Nucleophilic Aromatic Substitution: Addition–Elimination (SNAr)
Nucleophilic substitution in aromatic compounds is feasible in substrates bearing strong electron-withdrawing substituents positioned ortho or para to the leaving group. The reaction proceeds via two steps: the addition of the nucleophile and the elimination of the leaving group.
The reaction begins with an attack of the nucleophile on the carbon that holds the leaving group. This results in the delocalization of the π electrons over the ring carbons. The resonance interaction between the...
The reaction begins with an attack of the nucleophile on the carbon that holds the leaving group. This results in the delocalization of the π electrons over the ring carbons. The resonance interaction between the...
01:11Nucleophilic Aromatic Substitution: Elimination–Addition
Simple aryl halides do not react with nucleophiles. However, nucleophilic aromatic substitutions can be forced under certain conditions, such as high temperatures or strong bases. The mechanism of substitution under such conditions involves the highly unstable and reactive benzyne intermediate. Benzyne contains equivalent carbon centers at both ends of the triple bond, each of which is equally susceptible to nucleophilic attack. This 50–50 distribution of products is confirmed through isotopic...
01:13Carboxylic Acids to Esters: Acid-Catalyzed (Fischer) Esterification Mechanism
Carboxylic acids react with alcohols to yield esters via an acid-catalyzed condensation reaction called Fischer esterification. This is a nucleophilic acyl substitution reaction that proceeds via a tetrahedral intermediate, where a water molecule is eliminated as the leaving group.