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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.25942-0
2003-02-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/149/2/mic149451.html?itemId=/content/journal/micro/10.1099/mic.0.25942-0&mimeType=html&fmt=ahah

References

  1. Basnayake W. V. S., Birch R. G. 1995; A gene from Alcaligenes denitrificans that confers albicidin resistance by reversible antibiotic binding. Microbiology 141:551–560
    [Google Scholar]
  2. Birch R. G., Patil S. S. 1985a; Antibiotic and process for the production thereof. US Patent no. 4 525 354
    [Google Scholar]
  3. Birch R. G., Patil S. S. 1985b; Preliminary characterization of an antibiotic produced by Xanthomonas albilineans which inhibits DNA synthesis in Escherichia coli . J Gen Microbiol 131:1069–1075
    [Google Scholar]
  4. Birch R. G., Patil S. S. 1987; Evidence that an albicidin-like phytotoxin induces chlorosis in sugarcane leaf scald disease by blocking plastid DNA replication. Physiol Mol Plant Pathol 30:207–214
    [Google Scholar]
  5. Birch R. G, Pemberton J. M., Basnayake W. V. S. 1990; Stable albicidin resistance in Escherichia coli involves an altered outer-membrane nucleoside uptake system. J Gen Microbiol 136:51–58
    [Google Scholar]
  6. Gu J. Y, Yu G. Q, Zhu J. B., Shen S. J. 2000; The N-terminal domain of NifA determines the temperature sensitivity of NifA in Klebsiella pneumoniae and Enterobacter cloacae . Sci China Ser C Life Sci 43:8–15
    [Google Scholar]
  7. Huang G. Z, Zhang L. H., Birch R. G. 2000a; Analysis of the genes flanking xabB : a methyltransferase gene is involved in albicidin biosynthesis in Xanthomonas albilineans . Gene 255:327–333
    [Google Scholar]
  8. Huang G. Z, Zhang L. H., Birch R. G. 2000b; Albicidin antibiotic and phytotoxin biosynthesis in Xanthomonas albilineans requires a phosphopantetheinyl transferase gene. Gene 258:193–199
    [Google Scholar]
  9. Huang G. Z, Zhang L. H., Birch R. G. 2001; A multifunctional polyketide-peptide synthetase essential for albicidin biosynthesis by Xanthomonas albilineans . Microbiology 147:631–642
    [Google Scholar]
  10. Miles E. W. 1977; Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol 47:431–442
    [Google Scholar]
  11. Miller G. J., Ball E. H. 2001; Conformational change in the vinculin C-terminal depends on a critical histidine residue (His-906. J Biol Chem 276:28829–28834
    [Google Scholar]
  12. Monteiro R. A, Souza E. M, Funayama S, Yates M. G, Pedrosa F. O., Chubatsu L. S. 1999; Expression and functional analysis of an N-truncated NifA protein of Herbaspirillum seropedicae . FEBS Lett 447:283–286
    [Google Scholar]
  13. Ovadi J, Libor S., Elodi P. 1967; Spectrophotometric determination of histidine in proteins with diethyl pyrocarbonate. Biochim Biophys Acta 2:455–458
    [Google Scholar]
  14. Plasterer T. N. 2000; protean. Protein sequence analysis and prediction. Mol Biotechnol 16:117–125
    [Google Scholar]
  15. Rua J, Soler J, Busto F., de Arriaga D. 1995; The pH dependence and modification by diethyl pyrocarbonate of isocitrate lyase from Phycomyces blakesleeanus . Eur J Biochem 232:381–390
    [Google Scholar]
  16. Salazar J. C, Zuniga R, Lefimil C, Soll D., Orellana O. 2001; Conserved amino acids near the carboxy terminus of bacterial tyrosyl-tRNA synthesase are involved in tRNA and Tyr–AMP binding. FEBS Lett 491:257–260
    [Google Scholar]
  17. Sambrook J, Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  18. Souza E. M, Pedrosa F. O, Rigo L. U, Machado H. B., Yates M. G. 2000; Expression of the nifA gene of Herbaspirillum seropedicae : role of the NtrC and NifA binding sites and of the −24/−12 promoter element. Microbiology 146:1407–1418
    [Google Scholar]
  19. Walker M. J, Birch R. G., Pemberton J. M. 1988; Cloning and characterization of an albicidin resistance gene from Klebsiella oxytoca . Mol Microbiol 2:443–454
    [Google Scholar]
  20. Wiebe C. A, Dibattista E. R., Fliegel L. 2001; Functional role of polar amino acid residues in Na+/H+ exchangers. Biochem J 357:1–10
    [Google Scholar]
  21. Zhang L. H., Birch R. G. 1997; The gene for albicidin detoxification from Pantoea dispersa encodes an esterase and attenuates pathogenicity of Xanthomonas albilineans to sugarcane. Proc Natl Acad Sci U S A 94:9984–9989
    [Google Scholar]
  22. Zhang L. H, Xu J. L., Birch R. G. 1998a; Factors affecting biosynthesis by Xanthomonas albilineans of albicidin antibiotics and phytotoxins. J Appl Microbiol 85:1023–1028
    [Google Scholar]
  23. Zhang L. H, Xu J. L., Birch R. G. 1998b; High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca . Microbiology 144:555–559
    [Google Scholar]
  24. Zhang L. H, Xu J. L., Birch R. G. 1999; Engineered detoxification confers resistance against a pathogenic bacterium. Nat Biotechnol 17:1021–1024
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.25942-0
Loading
/content/journal/micro/10.1099/mic.0.25942-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error