title: An inserted α/β subdomain shapes the catalytic pocket of Lactobacillus johnsonii cinnamoyl esterase.
abstract: BACKGROUND
  Microbial enzymes produced in the gastrointestinal tract are primarily responsible for the release and biochemical transformation of absorbable bioactive monophenols. In the present work we described the crystal structure of LJ0536, a serine cinnamoyl esterase produced by the probiotic bacterium Lactobacillus johnsonii N6.2.
  
  
  RESULTS
  We crystallized LJ0536 in the apo form and in three substrate-bound complexes. The structure showed a canonical α/β fold characteristic of esterases, and the enzyme is dimeric. Two classical serine esterase motifs (GlyXSerXGly) can be recognized from the amino acid sequence, and the structure revealed that the catalytic triad of the enzyme is formed by Ser(106), His(225), and Asp(197), while the other motif is non-functional. In all substrate-bound complexes, the aromatic acyl group of the ester compound was bound in the deepest part of the catalytic pocket. The binding pocket also contained an unoccupied area that could accommodate larger ligands. The structure revealed a prominent inserted α/β subdomain of 54 amino acids, from which multiple contacts to the aromatic acyl groups of the substrates are made. Inserts of this size are seen in other esterases, but the secondary structure topology of this subdomain of LJ0536 is unique to this enzyme and its closest homolog (Est1E) in the Protein Databank.
  
  
  CONCLUSIONS
  The binding mechanism characterized (involving the inserted α/β subdomain) clearly differentiates LJ0536 from enzymes with similar activity of a fungal origin. The structural features herein described together with the activity profile of LJ0536 suggest that this enzyme should be clustered in a new group of bacterial cinnamoyl esterases.
authors.affiliation: Department of Microbiology and Cell Science, Genetics Institute, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Kin-Kwan
authors.initials: KK
authors.lastname: Lai
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Stogios
authors.forename: Clara
authors.initials: C
authors.lastname: Vu
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Xu
authors.forename: Hong
authors.initials: H
authors.lastname: Cui
authors.forename: Sara
authors.initials: S
authors.lastname: Molloy
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.forename: Alexander
authors.initials: A
authors.lastname: Yakunin
authors.forename: Claudio F
authors.initials: CF
authors.lastname: Gonzalez
mesh_terms: D000409:Alanine / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D008958:Models, Molecular; D016254:Mutagenesis, Insertional / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D050505:Mutant Proteins; D012694:Serine; D002265:Carboxylic Ester Hydrolases; D000409:Alanine