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Kinetic and substrate complex characterization of RamA, a corrinoid protein reductive activase from Methanosarcina barkeri

Journal Article · · FEMS Microbiology Letters (Online)
 [1];  [2];  [3]
  1. Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA; OSTI
  2. Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA
  3. Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA
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ABSTRACT

In microbial corrinoid-dependent methyltransferase systems, adventitious Co(I)-corrinoid oxidation halts catalysis and necessitates repair by ATP-dependent reductive activases. RamA, an activase with a C-terminal ferredoxin domain with two [4Fe-4S] clusters from methanogenic archaea, has been far less studied than the bacterial activases bearing an N-terminal ferredoxin domain with one [2Fe-2S] cluster. These differences suggest RamA might prove to have other distinctive characteristics. Here, we examine RamA kinetics and the stoichiometry of the corrinoid protein:RamA complex. Like bacterial activases, K+ stimulates RamA. Potassium stimulation had been questioned due to differences in the primary structure of bacterial and methanogen activases. Unlike one bacterial activase, ATP is not inhibitory allowing the first determination of apparent kinetic parameters for any corrinoid activase. Unlike bacterial activases, a single RamA monomer complexes a single corrinoid protein monomer. Alanine replacement of a RamA serine residue corresponding to the serine of one bacterial activase which ligates the corrinoid cobalt during complex formation led to only moderate changes in the kinetics of RamA. These results reveal new differences in the two types of corrinoid activases, and provide direct evidence for the proposal that corrinoid activases act as catalytic monomers, unlike other enzymes that couple ATP hydrolysis to difficult reductions.

Research Organization:
The Ohio State Univ., Columbus, OH (United States)
Sponsoring Organization:
USDOE Office of Science (SC)
DOE Contract Number:
FG02-91ER20042;
OSTI ID:
1804034
Journal Information:
FEMS Microbiology Letters (Online), Journal Name: FEMS Microbiology Letters (Online) Journal Issue: 17 Vol. 367; ISSN 1574-6968
Publisher:
Federation of European Microbiological Societies
Country of Publication:
United States
Language:
English

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