A multi-faceted analysis of RutD reveals a novel family of α/β hydrolases

Aleksandra A. Knapik, Janusz J. Petkowski, Zbyszek Otwinowski, Marcin T. Cymborowski, David R. Cooper, Karolina A. Majorek, Maksymilian Chruszcz, Wanda M. Krajewska, Wladek Minor

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


The rut pathway of pyrimidine catabolism is a novel pathway that allows pyrimidine bases to serve as the sole nitrogen source in suboptimal temperatures. The rut operon in E. coli evaded detection until 2006, yet consists of seven proteins named RutA, RutB, etc. through RutG. The operon is comprised of a pyrimidine transporter and six enzymes that cleave and further process the uracil ring. Herein, we report the structure of RutD, a member of the α/β hydrolase superfamily, which is proposed to enhance the rate of hydrolysis of aminoacrylate, a toxic side product of uracil degradation, to malonic semialdehyde. Although this reaction will occur spontaneously in water, the toxicity of aminoacrylate necessitates catalysis by RutD for efficient growth with uracil as a nitrogen source. RutD has a novel and conserved arrangement of residues corresponding to the α/β hydrolase active site, where the nucleophile's spatial position occupied by Ser, Cys, or Asp of the canonical catalytic triad is replaced by histidine. We have used a combination of crystallographic structure determination, modeling and bioinformatics, to propose a novel mechanism for this enzyme. This approach also revealed that RutD represents a previously undescribed family within the α/β hydrolases. We compare and contrast RutD with PcaD, which is the closest structural homolog to RutD. PcaD is a 3-oxoadipate-enol-lactonase with a classic arrangement of residues in the active site. We have modeled a substrate in the PcaD active site and proposed a reaction mechanism. Proteins 2012;.

Original languageEnglish (US)
Pages (from-to)2359-2368
Number of pages10
JournalProteins: Structure, Function and Bioinformatics
Issue number10
StatePublished - Oct 2012


  • 3-oxoadipate enol-lactone
  • 3-oxoadipate-enol-lactonase
  • Aminoacrylate
  • E. coli
  • PcaD
  • Rut pathway of pyrimidine degradation
  • RutD
  • α/β hydrolases

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology


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