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Research article

References

  1. Abou Nader M. 2012. Directed evolution of cyanide degrading enzymes. Doctoral dissertation. Texas A&M University, TX.
  2. Fisher FB, Brown JS. 1952. Colorimetric determination of cyanide in stack gas and waste water. Anal. Chem. 24: 14401444.
    CrossRef
  3. Jandhyala D, Berman M, Meyers PR, Sewell BT, Willson RC, Benedik MJ. 2003. CynD, the cyanide dihydratase from Bacillus pumilus: gene cloning and structural studies. Appl. Environ. Microbiol. 69: 4794-4805.
    Pubmed PMC CrossRef
  4. Kimani SW, Agarkar VB, Cowan DA, Sayed MF, Sewell BT. 2007. S tructure o f an a liph atic a midase f rom Geobacillus pallidus RAPc8. Acta Crystallogr. D Biol. Crystallogr. 63: 10481058.
    Pubmed CrossRef
  5. Kumaran D, Eswaramoorthy S, Gerchman SE, Kycia H, Studier FW, Swaminathan S. 2003. Crystal structure of a putative CN hydrolase from yeast. Proteins 52: 283-291.
    Pubmed CrossRef
  6. Lundgren S, Lohkamp B, Andersen B, Piskur J, Dobritzsch D. 2008. The crystal structure of beta-alanine synthase from Drosophila melanogaster r eveals a h omooctameric h elical turn-like assembly. J. Mol. Biol. 377: 1544-1559.
    Pubmed CrossRef
  7. Meyers PR, Rawlings DE, Woods DR, Lindsey GG. 1993. Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1. J. Bacteriol. 175: 6105-6112.
    Pubmed PMC CrossRef
  8. Muezzinoglu A. 2003. A review of environmental considerations on gold mining and production. Crit. Rev. Environ. Sci. Technol. 33: 45-71.
    CrossRef
  9. Nagasawa T, Wieser M, Nakamura T, Iwahara H, Yoshida T, Gekko K. 2000. Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association. Eur. J. Biochem. 267: 138-144.
    Pubmed CrossRef
  10. Pace HC, Brenner C. 2001. The nitrilase superfamily:classification, structure and function. Genome Biol. 2:reviews0001.1-reviews0001.9.
  11. Pace HC, Hodawadekar SC, Draganescu A, Huang J, Bieganowski P, Pekarsky Y, et al. 2000. Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers. Curr. Biol. 10: 907-917.
    CrossRef
  12. Park JM, Mulelu A, Sewell BT, Benedik MJ. 2015. Probing an interfacial surface in the cyanide dihydratase from Bacillus pumilus, a spiral forming nitrilase. Front. Microbiol. 6: 1479.
    Pubmed
  13. Sewell BT, Berman MN, Meyers PR, Jandhyala D, Benedik MJ. 2003. The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral. Structure 11: 1413-1422.
    Pubmed CrossRef
  14. Sewell BT, Thuku RN, Zhang X, Benedik MJ. 2005. Oligomeric structure of nitrilases: effect of mutating interfacial residues on activity. Ann. NY Acad. Sci. 1056: 153-159.
    Pubmed CrossRef
  15. Thuku RN, Brady D, Benedik MJ, Sewell BT. 2009. Microbial nitrilases: versatile, spiral forming, industrial enzymes. J. Appl. Microbiol. 106: 703-727.
    Pubmed CrossRef
  16. Th uku RN, Weber BW, V arsani A , Sewell B T. 2 007. P osttranslational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form. FEBS J. 274: 2099-2108.
    Pubmed CrossRef
  17. Wang L, Watermeyer JM, Mulelu AE, Sewell BT, Benedik MJ. 2012. Engineering pH-tolerant mutants of a cyanide dihydratase. Appl. Microbiol. Biotechnol. 94: 131-140.
    Pubmed CrossRef

Article

Research article

J. Microbiol. Biotechnol. 2016; 26(12): 2179-2183

Published online December 28, 2016 https://doi.org/10.4014/jmb.1606.06035

Copyright © The Korean Society for Microbiology and Biotechnology.

Residue Y70 of the Nitrilase Cyanide Dihydratase from Bacillus pumilus Is Critical for Formation and Activity of the Spiral Oligomer

Jason M. Park 1, Christian M. Ponder 1, B. Trevor Sewell 2 and Michael J. Benedik 1*

1Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA, 2Structural Biology Research Unit, Institute for Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town 7925, South Africa

Received: June 15, 2016; Accepted: August 25, 2016

Abstract

Nitrilases pose attractive alternatives to the chemical hydrolysis of nitrile compounds. The
activity of bacterial nitrilases towards substrate is intimately tied to the formation of large
spiral-shaped oligomers. In the nitrilase CynD (cyanide dihydratase) from Bacillus pumilus,
mutations in a predicted oligomeric surface region altered its oligomerization and reduced its
activity. One mutant, CynD Y70C, retained uniform oligomer formation however it was
inactive, unlike all other inactive mutants throughout that region all of which significantly
perturbed oligomer formation. It was hypothesized that Y70 is playing an additional role
necessary for CynD activity beyond influencing oligomerization. Here, we performed
saturation mutagenesis at residue 70 and demonstrated that only tyrosine or phenylalanine is
permissible for CynD activity. Furthermore, we show that other residues at this position are
not only inactive, but have altered or disrupted oligomer conformations. These results suggest
that Y70’s essential role in activity is independent of its role in the formation of the spiral
oligomer.

Keywords: nitrilase, cyanide dihydratase, quaternary structure, cyanide, oligomerization surface, bioremediation

References

  1. Abou Nader M. 2012. Directed evolution of cyanide degrading enzymes. Doctoral dissertation. Texas A&M University, TX.
  2. Fisher FB, Brown JS. 1952. Colorimetric determination of cyanide in stack gas and waste water. Anal. Chem. 24: 14401444.
    CrossRef
  3. Jandhyala D, Berman M, Meyers PR, Sewell BT, Willson RC, Benedik MJ. 2003. CynD, the cyanide dihydratase from Bacillus pumilus: gene cloning and structural studies. Appl. Environ. Microbiol. 69: 4794-4805.
    Pubmed KoreaMed CrossRef
  4. Kimani SW, Agarkar VB, Cowan DA, Sayed MF, Sewell BT. 2007. S tructure o f an a liph atic a midase f rom Geobacillus pallidus RAPc8. Acta Crystallogr. D Biol. Crystallogr. 63: 10481058.
    Pubmed CrossRef
  5. Kumaran D, Eswaramoorthy S, Gerchman SE, Kycia H, Studier FW, Swaminathan S. 2003. Crystal structure of a putative CN hydrolase from yeast. Proteins 52: 283-291.
    Pubmed CrossRef
  6. Lundgren S, Lohkamp B, Andersen B, Piskur J, Dobritzsch D. 2008. The crystal structure of beta-alanine synthase from Drosophila melanogaster r eveals a h omooctameric h elical turn-like assembly. J. Mol. Biol. 377: 1544-1559.
    Pubmed CrossRef
  7. Meyers PR, Rawlings DE, Woods DR, Lindsey GG. 1993. Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1. J. Bacteriol. 175: 6105-6112.
    Pubmed KoreaMed CrossRef
  8. Muezzinoglu A. 2003. A review of environmental considerations on gold mining and production. Crit. Rev. Environ. Sci. Technol. 33: 45-71.
    CrossRef
  9. Nagasawa T, Wieser M, Nakamura T, Iwahara H, Yoshida T, Gekko K. 2000. Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association. Eur. J. Biochem. 267: 138-144.
    Pubmed CrossRef
  10. Pace HC, Brenner C. 2001. The nitrilase superfamily:classification, structure and function. Genome Biol. 2:reviews0001.1-reviews0001.9.
  11. Pace HC, Hodawadekar SC, Draganescu A, Huang J, Bieganowski P, Pekarsky Y, et al. 2000. Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers. Curr. Biol. 10: 907-917.
    CrossRef
  12. Park JM, Mulelu A, Sewell BT, Benedik MJ. 2015. Probing an interfacial surface in the cyanide dihydratase from Bacillus pumilus, a spiral forming nitrilase. Front. Microbiol. 6: 1479.
    Pubmed
  13. Sewell BT, Berman MN, Meyers PR, Jandhyala D, Benedik MJ. 2003. The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral. Structure 11: 1413-1422.
    Pubmed CrossRef
  14. Sewell BT, Thuku RN, Zhang X, Benedik MJ. 2005. Oligomeric structure of nitrilases: effect of mutating interfacial residues on activity. Ann. NY Acad. Sci. 1056: 153-159.
    Pubmed CrossRef
  15. Thuku RN, Brady D, Benedik MJ, Sewell BT. 2009. Microbial nitrilases: versatile, spiral forming, industrial enzymes. J. Appl. Microbiol. 106: 703-727.
    Pubmed CrossRef
  16. Th uku RN, Weber BW, V arsani A , Sewell B T. 2 007. P osttranslational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form. FEBS J. 274: 2099-2108.
    Pubmed CrossRef
  17. Wang L, Watermeyer JM, Mulelu AE, Sewell BT, Benedik MJ. 2012. Engineering pH-tolerant mutants of a cyanide dihydratase. Appl. Microbiol. Biotechnol. 94: 131-140.
    Pubmed CrossRef