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The PrpF protein of Shewanella oneidensis MR-1 catalyzes the isomerization of 2-methyl-cis-aconitate during the catabolism of propionate via the AcnD-dependent 2-methylcitric acid cycle

Journal Article · · PLoS ONE
 [1];  [2];  [3];  [2];  [4];  [5]
  1. The Research Institute at Nationwide Children’s Hospital, Columbus, OH (United States); Univ. of Wisconsin, Madison, WI (United States)
  2. Univ. of Wisconsin, Madison, WI (United States)
  3. Monsanto Vegetable Seeds, Woodland, CA (United States); Univ. of Wisconsin, Madison, WI (United States)
  4. Univ. of Georgia, Athens, GA (United States)
  5. Univ. Nova de Lisboa Inst. de Tecnologia Quimica e Biologica (Portugal)
+ Show Author Affiliations
The 2-methylcitric acid cycle (2-MCC) is a common route of propionate catabolism in microorganisms. In Salmonella enterica, the prpBCDE operon encodes most of the 2-MCC enzymes. In other organisms, e.g., Shewanella oneidensis MR-1, two genes, acnD and prpF replace prpD, which encodes 2-methylcitrate dehydratase. We showed that together, S. oneidensis AcnD and PrpF (SoAcnD, SoPrpF) compensated for the absence of PrpD in a S. enterica prpD strain. We also showed that SoAcnD had 2-methylcitrate dehydratase activity and that PrpF has aconitate isomerase activity. Here we report in vitro evidence that the product of the SoAcnD reaction is an isomer of 2-methyl-cis-aconitate (2-MCA], the product of the SePrpD reaction. We show that the SoPrpF protein isomerizes the product of the AcnD reaction into the PrpD product (2-MCA], a known substrate of the housekeeping aconitase (AcnB]. Given that SoPrpF is an isomerase, that SoAcnD is a dehydratase, and the results from in vivo and in vitro experiments reported here, it is likely that 4-methylaconitate is the product of the AcnD enzyme. Results from in vivo studies using a S. enterica prpD strain show that SoPrpF variants with substitutions of residues K73 or C107 failed to support growth with propionate as the sole source of carbon and energy. High-resolution (1.22 Å) three-dimensional crystal structures of PrpFK73E in complex with trans-aconitate or malonate provide insights into the mechanism of catalysis of the wild-type protein.
Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
National Inst. of Allergy and Infectious Diseases; National Inst. of General Medical Sciences
OSTI ID:
1498362
Journal Information:
PLoS ONE, Journal Name: PLoS ONE Journal Issue: 11 Vol. 12; ISSN 1932-6203
Publisher:
Public Library of ScienceCopyright Statement
Country of Publication:
United States
Language:
ENGLISH

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Salmonella enterica
catabolism
crystal structure
dehydration (medicine)
high performance liquid chromatography
isomerases
operons
propionates