Computer-assisted docking of flavodoxin with the ATP:Co(I)rrinoid adenosyltransferase (CobA) enzyme reveals residues critical for protein-protein interactions but not for catalysis

Nicole R. Buan, Jorge C. Escalante-Semerena

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17 Citations (Scopus)

Abstract

The activity of the housekeeping ATP:co(I)rrinoid adenosyl-transferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobA and FldA interact, a computer model of a CobA·FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(I)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA·FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4%. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ.

Original languageEnglish (US)
Pages (from-to)40948-40956
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number49
DOIs
StatePublished - Dec 9 2005

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Flavodoxin
Catalysis
Corrinoids
Salvaging
Housekeeping
Mutagenesis
Salmonella
Salmonella enterica
Substrates
Enzymes
Vitamin B 12
Transferases
Site-Directed Mutagenesis
Computer Simulation
Catalytic Domain
Bacteria
Proteins
Adenosine Triphosphate
Electrons
Amino Acids

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Computer-assisted docking of flavodoxin with the ATP:Co(I)rrinoid adenosyltransferase (CobA) enzyme reveals residues critical for protein-protein interactions but not for catalysis",
abstract = "The activity of the housekeeping ATP:co(I)rrinoid adenosyl-transferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobA and FldA interact, a computer model of a CobA·FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(I)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA·FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4{\%}. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ.",
author = "Buan, {Nicole R.} and Escalante-Semerena, {Jorge C.}",
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T1 - Computer-assisted docking of flavodoxin with the ATP:Co(I)rrinoid adenosyltransferase (CobA) enzyme reveals residues critical for protein-protein interactions but not for catalysis

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AU - Escalante-Semerena, Jorge C.

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N2 - The activity of the housekeeping ATP:co(I)rrinoid adenosyl-transferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobA and FldA interact, a computer model of a CobA·FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(I)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA·FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4%. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ.

AB - The activity of the housekeeping ATP:co(I)rrinoid adenosyl-transferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobA and FldA interact, a computer model of a CobA·FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(I)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA·FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4%. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ.

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