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
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ABSTRACT |
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The GenBank accession number for the albA gene sequence from Klebsiella oxytoca ATCC 13182T is AF525464.
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INTRODUCTION |
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Albicidins are bactericidal at nanomolar concentrations against a range of Gram-positive and Gram-negative bacteria (Birch & Patil, 1985a). The exquisite sensitivity is due to illicit uptake of albicidins through a nucleoside uptake channel (Birch et al., 1990
). Inhibition of DNA replication in bacteria and plastids is the primary mechanism of action (Birch & Patil, 1985b
, 1987
). Several albicidin-resistance mechanisms have been identified, including albicidin-binding proteins (Walker et al., 1988
; Basnayake & Birch, 1995
) and an esterase which hydrolyses and detoxifies albicidin (Zhang & Birch, 1997
). Expression of the albicidin esterase, either in the pathogen or in transgenic sugarcane, prevents development of chlorotic disease symptoms and systemic invasion in inoculated plants (Zhang & Birch, 1997
; Zhang et al., 1999
).
The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin with strong affinity and selectivity. This is of interest as a potential phytotoxin- and disease-resistance mechanism, and as a novel ligandmatrix combination for biotechnological applications (Walker et al., 1988; Zhang et al., 1998b
). Kinetic and stoichiometric analyses indicate the presence of a single high-affinity binding site with a dissociation constant of 6·4x10-8 M in the AlbA protein (Zhang et al., 1998b
). Albicidin-binding capacity decreases by 30 % when pH is shifted from 6 to 4, which is the range for side-chain ionization of histidine residues.
To assess the role of histidine residues in the AlbAalbicidin interaction, we first cloned and resequenced the albA gene from K. oxytoca ATCC 13182T, and we report here the revised albA DNA and peptide sequences. We show, by stoichiometric analysis and site-directed mutagenesis, that His125 is uniquely involved in albicidin binding.
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METHODS |
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Protein sequence analysis and prediction.
Protein sequence analysis and secondary structure prediction were performed using the PROTEAN module in DNASTAR (Plasterer, 2000).
Site-directed mutagenesis.
Site-directed mutagenesis of albA in pGST-AlbA was performed by using a QuikChange Site-Directed Mutagenesis Kit according to the manufacturer's recommendations (Stratagene). The four histidines of AlbA were converted to glycine or other amino acids using the primers listed in Table 2. Each mutated clone was sequenced to verify the desired mutation and assayed for albicidin-binding activity.
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AlbAalbicidin binding assay.
AlbA variants (0·77 µM) in TMM buffer (10 mM Tris/HCl, pH 7·0; 10 mM MgCl2; 2 mM 2-mercaptoethanol) were mixed with albicidin at concentrations ranging from 0·12 to 1·17 µM, then incubated at 25 °C for 5 min before a quantitative assay of free albicidin as described previously (Zhang et al., 1998b). Briefly, 100 µl of a fresh E. coli DH5
culture, which was adjusted to an OD600 value of 1·8, were added to 5 ml of 1 % (w/v) agarose at 50 °C. The mixture was quickly added to a plate containing 20 ml of LB agar. After solidification, wells 4 mm in diameter were punched in the agar, 20 µl of the test solution was added to each well and the plate was incubated at 37 °C overnight. Free albicidin in the test solution was calculated using the formula albicidin (ng ml-1)=4·576e(0·315w), where w is the width (in mm) of the zone of growth inhibition surrounding the well. This formula is derived from the linear portion of an albicidin dose-response plot under the same assay conditions (Zhang et al., 1998a
). Bound albicidin was calculated by subtracting the amount of free albicidin from the total albicidin added to the reaction mixture.
Inactivation of AlbA by diethyl pyrocarbonate (DEPC).
DEPC was stored desiccated at 4 °C to minimize decomposition by hydrolysis. The stock solution of DEPC was freshly prepared by diluting DEPC with cold absolute ethanol (1 : 19, v/v). Buffered DEPC solutions were prepared immediately before use in 0·1 M sodium phosphate buffer (pH 6·5) and kept on ice. The reaction mixture was prepared by adding 0·1 ml of buffered DEPC solution to 0·9 ml of AlbA in the same buffer to obtain the final concentrations specified below. The mixtures were incubated at 25 °C, and aliquots were taken at the times specified for photometry and assays of albicidin-binding activity.
UV differential spectra and stoichiometry of histidine modification.
UV absorbance spectra were recorded for purified AlbA (3 mg ml-1), before and after treatment with 13·8 mM DEPC for 10 min at 25 °C. For kinetics and stoichiometry, purified AlbA (0·2 mg ml-1) was treated with DEPC concentrations ranging from 0·05 to 1·5 mM. The extent of histidine residue modification was calculated from the absorbance change at 242 nm, the absorption coefficient (3200 M-1 cm-1) of N-carbethoxyhistidine (Ovadi et al., 1967; Miles, 1977
) and the molar concentration of AlbA (Mr=25 852 Da). Immediately after the absorbance measurement, albicidin was added (100 ng ml-1) and the residual albicidin-binding activity was determined by bioassay.
Substrate protection against AlbA modification by DEPC.
To obtain the specified albicidin to AlbA molar ratios, 40 µl of an albicidin stock in 50 % (v/v) methanol was added to 860 µl of AlbA solution (0·1 mg ml-1), and incubated at 25 °C for 5 min before the addition of 100 µl of DEPC solution (10 mM). The final concentration of methanol in the reaction mixture was 2 %, which did not affect albicidin binding or DEPC modification. The stoichiometry of N-carbethoxyhistidine residue formation was determined as described above.
Hydroxylamine treatment of inactivated enzyme.
After 10 min incubation of AlbA at 25 °C with 0·25 mM DEPC, hydroxylamine was added to a final concentration of 100 mM. The mixtures were then incubated at 4 °C for 40 min. Absorbance at 242 nm and albicidin-binding activity were determined before and after hydroxylamine treatment.
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RESULTS |
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The predicted AlbA peptide has 221 aa residues, an isoelectric point at 4·9 and four histidine residues (His78, His125, His141 and His189). It shows a low level of sequence similarity to the DNA-binding proteins encoded by ntrC from Acidithiobacillus ferrooxidans (Salazar et al., 2001) and nifA from several bacterial species (Monteiro et al., 1999
; Gu et al., 2000
; Souza et al., 2000
). There is no significant similarity beyond that previously noted at the N terminus between AlbA and AlbB from Alcaligenes denitrificans, which also encodes an albicidin-binding protein (Basnayake & Birch, 1995
).
Effect of DEPC on albicidin-binding activity of AlbA
We used DEPC, a histidine-specific reagent, to probe the role of the histidine residues of AlbA in binding to albicidin, as a previous study had suggested that histidine residues could be involved in the proteinligand interaction (Zhang et al., 1998b). After incubation of AlbA with 0·05 mM DEPC for 13 min, about 55 % of the albicidin-binding activity was lost. In 1·5 mM DEPC, about 95 % of the binding activity was lost within 5 min (Fig. 1
a).
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Substrate protection against histidine residue modification by DEPC
The AlbAalbicidin complex is very stable, with albicidin released only under protein denaturing conditions (Zhang et al., 1998b). When albicidin was mixed with AlbA at different molar ratios before addition of DEPC, the percentage of histidine residues alkylated was inversely proportional to the albicidin to AlbA molar ratio (Fig. 1d
). Thus, bound albicidin blocked the reaction between DEPC and the active His residue.
Stoichiometry of inactivation
AlbA was treated with DEPC concentrations ranging from 0·05 to 1·5 mM for 5 min. The reaction was stopped by adding albicidin. The relationship between the extent of histidine residue modification and the residue-binding activity of AlbA was calculated from the change in the OD240 value and bioassay of binding activity of DEPC-treated AlbA. There are four histidine residues in AlbA; the stoichiometry data show that only one histidine residue has been modified by DEPC (Fig. 1c). There was no further increase in the number of histidine residues alkylated at higher DEPC concentrations or extended incubation times. Albicidin-binding activity decreased linearly with modification of the histidine residue (Fig. 1c
).
Prediction of the active histidine residue in AlbA
We assumed that the three histidine residues in AlbA that were not alkylated by DEPC might be located in regions whose structural features prevent access by DEPC. Secondary structure predictions for AlbA from the GARNIERROBSON program indicate that His78, His141 and His189 are located in -helix regions, whereas His125 lies at the immediate front of a turn region (Fig. 2
). The EMINI program indicates that His125 is most likely exposed, with a surface probability (SP) index of 2·4. The other histidine residues have SP indices of less than 1·4 (Fig. 2
). Thus, His125 is likely to be the active histidine residue of AlbA that interacts with DEPC and albicidin.
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DISCUSSION |
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Histidine residues are involved in substrate binding by many enzymes. The active histidine residue may act as a proton acceptor or donor in proteinligand interactions, chargerelay interactions between amino acids at the active site, conformational changes associated with substrate binding or oligomerization of protein chains (Miller & Ball, 2001; Wiebe et al., 2001
).
In AlbA, the deletion of His125 abolished albicidin-binding activity. Replacement of His125 with non-polar amino acids (glycine, alanine or leucine) caused about 30 % decrease in albicidin-binding activity (Fig. 3). Lowering the pH to 4, which results in protonation of histidine (pKa=6·0), also decreased albicidin-binding activity by about 30 % (Zhang et al., 1998b
). Replacement of His125 with amino acids with similar properties (arginine or tyrosine) had little effect on the albicidin-binding capacity of AlbA. These results could indicate the importance of a proton donor at residue 125 for the conformational change of AlbA to form the albicidin-binding region, or for an electrostatic interaction between AlbA and albicidin.
Using the GARNIERROBSON program in the PROTEAN module, deletion of His125 was predicted to greatly shorten the turn region between the adjoining -helices (Fig. 2
). DEPC treatment converting a single histidine residue to N-carbethoxyhistidine resulted in an almost-complete loss of albicidin-binding activity (Fig. 1c
). The bulky side chain of N-carbethoxyhistidine (Fig. 3
) could prevent proper entry of albicidin to the substrate-binding cleft, or hinder a conformational change of AlbA required for albicidin binding.
The full chemical structure of albicidin has not yet been elucidated. NMR and MS analyses on the major antibiotic component produced in rich culture media indicates a structure with 38 carbon atoms including three to four aromatic rings, a probable O-methyl tyrosine and at least one COOH group (Birch & Patil, 1985a; Huang et al., 2001
). Biosynthesis involves a multifunctional polyketide-peptide synthetase (Huang et al., 2001
), and albicidin is cleaved by the AlbD esterase (Zhang & Birch, 1997
). Of the four amino acids in the predicted turn region following His125 of AlbA, three have polar side chains (Tyr, Asp and Arg) that could interact with the COOH group or the OH group of O-methyl tyrosine of albicidin through H-bonding.
Computer-based structural analysis is well established for the prediction of active residues in proteins with conserved structural features and sequence motifs. Our results show that computer-based predictions of protein secondary structure and amino acid surface probability can be useful for indicating the residues that are likely to interact with a ligand, even in proteins without evident homology to known structural domains.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 August 2002;
revised 21 October 2002;
accepted 30 October 2002.
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