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Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

¹ Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609
² Department of Botany, The University of Queensland, Brisbane QLD 4072, Australia
³ Department of Biological Sciences, The National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260

Correspondence
Lian-Hui Zhang
lianhui{at}imcb.a-star.edu.sg

The albA gene from Klebsiella oxytoca 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 albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K. oxytoca ATCC 13182^T to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His₇₈, His₁₂₅, His₁₄₁ and His₁₈₉) 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 N-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 His₁₂₅ might be the residue required for albicidin binding. Mutation of His₁₂₅ to either alanine or leucine resulted in about 32 % loss of binding activity, and deletion of His₁₂₅ totally abolished binding activity. Mutation of His₁₂₅ to arginine and tyrosine had no effect. These results indicate that His₁₂₅ plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

The GenBank accession number for the albA gene sequence from Klebsiella oxytoca ATCC 13182^T is AF525464.

This article has been cited by other articles:

				L.-X. Weng, L.-H. Wang, J.-L. Xu, J.-E. Wu, Q. Li, and L.-H. Zhang Molecular and Conformational Basis of a Specific and High-Affinity Interaction between AlbA and Albicidin Phytotoxin Appl. Envir. Microbiol., March 1, 2005; 71(3): 1445 - 1452. [Abstract] [Full Text] [PDF]