1887

Abstract

The gene from encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30 %, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the coding region revealed sequence discrepancies with the sequence reported previously, which were probably due to sequencing errors. The gene was subsequently cloned from ATCC 13182 to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His, His, His and His) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95 %. DEPC treatment increased absorbance at 240–244 nm by an amount indicating conversion to -carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that His might be the residue required for albicidin binding. Mutation of His to either alanine or leucine resulted in about 32 % loss of binding activity, and deletion of His totally abolished binding activity. Mutation of His to arginine and tyrosine had no effect. These results indicate that His plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

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2003-02-01
2020-04-09
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