Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics.
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 (His(78), His(125), His(141) and His(189)) 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 His(125) might be the residue required for albicidin binding. Mutation of His(125) to either alanine or leucine resulted in about 32 % loss of binding activity, and deletion of His(125) totally abolished binding activity. Mutation of His(125) to arginine and tyrosine had no effect. These results indicate that His(125) plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.
Weng
Li-Xing
LX
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609.
Xu
Jin-Ling
JL
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609.
Li
Qi
Q
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609.
Birch
Robert G
RG
Department of Botany, The University of Queensland, Brisbane QLD 4072, Australia.
Zhang
Lian-Hui
LH
Department of Biological Sciences, The National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260.|Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609.
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017353:Gene Deletion; D006639:Histidine; D041121:Klebsiella oxytoca / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009930:Organic Chemicals; D017422:Sequence Analysis, DNA; D014974:Xanthomonas
C111474:AlbA protein, Klebsiella oxytoca; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D009930:Organic Chemicals; D006639:Histidine; C046156:albicidin