Structural basis of the broad substrate tolerance of the antibody 7B9-catalyzed hydrolysis of p-nitrobenzyl esters

Bioorganic & Medicinal Chemistry
Naoki MiyamotoI Fujii

Abstract

Catalytic antibody 7B9, which was elicited against p-nitrobenzyl phosphonate transition-state analogue (TSA) 1, hydrolyzes a wide range of p-nitrobenzyl monoesters and thus shows broad substrate tolerance. To reveal the molecular basis of this substrate tolerance, the 7B9 Fab fragment complexed with p-nitrobenzyl ethylphosphonate 2 was crystallized and the three-dimensional structure was determined. The crystal structure showed that the strongly antigenic p-nitrobenzyl moiety occupied a relatively shallow antigen-combining site and therefore the alkyl moiety was located outside the pocket. These results support the observed broad substrate tolerance of 7B9 and help rationalize how 7B9 can catalyze various p-nitrobenzyl ester derivatives. The crystal structure also showed that three amino acid residues (AsnH33, SerH95, and ArgL96) were placed in key positions to form hydrogen bonds with the phosphonate oxygens of the transitions-state analogue. In addition, the role of these amino acid residues was examined by site-directed mutagenesis to alanine: all mutants (AsnH33Ala, SerH95Ala, and ArgL96Ala) showed no detectable catalytic activity. Coupling the findings from our structural studies with these mutagenesis results clarified th...Continue Reading

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