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Title: Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined tomore » be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less
Authors:
 [1] ;  [1] ;  [2] ;  [3] ;  [4] ;  [3] ;  [5] ;  [6] ;  [7]
  1. Univ. of Missouri, Columbia, MO (United States). Biochemistry Division
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
  3. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Biochemistry and Cellular and Molecular Biology
  4. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Biochemistry and Cellular and Molecular Biology; Univ. of Tennessee, Knoxville, TN (United States). Dept. of of Microbiology
  5. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Biochemistry and Cellular and Molecular Biology; Univ. of Tennessee, Knoxville, TN (United States). Dept. of of Microbiology
  6. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Univ. of Tennessee, Knoxville, TN (United States). Dept. of Microbiology
  7. Univ. of Missouri, Columbia, MO (United States). Biochemistry Division; Univ. of Missouri, Columbia, MO (United States). Molecular Microbiology and Immunology
Publication Date:
OSTI Identifier:
1265498
Grant/Contract Number:
AC05-00OR22725
Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 81; Journal Issue: 9; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Research Org:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY mercury; mercury SFA; methylmercury; Hg; MeHg