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Project period : 2010 - 2012

Frank Raushel, Texas A&M


  • primarily hydrolytic cleavage of carbon-hetero atom bonds through H2O activation and substrate polarization via a mononuclear or binuclear metal center


  • distorted (β/α)8 barrel fold

Challenges for Function Assignment

  • shallow understanding of bacterial metabolism results in incomplete virtual and real libararies for computational and experimental screening
  • cryptic physiological functions due to roles in novel metabolism

Value to Integrated Strategy

  • serves as a paradigm for functional assignment through an integrated bioinformatic-computational-crystallographic approach
  • requires advances in bioinformatic methods to deal with massive and complex sequences sets
  • forces assessment of current understanding of bacterial metabolism

Conservation of both tertiary structure and active site architecture in the x-ray crystal structures of phosphotriesterase, urease, and adenosine deaminase led Holm and Sander to the discovery of the amidohydrolase (AH) superfamily.  While the majority of reactions in the AH superfamily are hydrolytic cleavages of amide or ester bonds, other members carry out deamination, decarboxylation, isomerization, hydration, or retroaldol cleavage totaling over 40 unique reactions. AH superfamily members have been discovered in every sequenced organism and >36,000 proteins have been identified to date representing ~0.5% of the protein universe.  This prevalence is easily understood given the functional diversity of the AH superfamily which includes roles in the metabolism of amino acids, carbohydrates, nucleic acids, and organophosphate esters.


Figure AH1. Active site of phosphotriesterase (PTE) highlighting conserved residues binding the α and β metals (black). Residues involved in substrate specificity and gate-keeping (orange) are found in loops extending from the β-strands of the (α/β)8 barrel. Other AH superfamily members contain only one metal in either the α or β site (see text).


Superfamily members are identified by the presence of six conserved active site residues that form binding sites for one or two Mn2+, Fe2+, Zn2+, or Ni2+ cations (Figure AH1).  Metal sites are termed α or β and may be mononuclear (occupying either the α or β site) or binuclear (occupying both the α and β sites).  Although conservation of the distorted (β/α)8 barrel fold indicates evolution from a common ancestor (Figure AH2), members within the AH superfamily share little overall sequence identity besides the salient active site residues.  This diversity is especially pronounced in loop regions that vary greatly in sequence, length, and secondary structure and are found at the C-terminal ends of the β-strands.  In some cases these loops serve gate keeping functions by undergoing conformational changes which enable substrate recognition and binding in the active site.


Figure AH2. Comparison of the (α/β)8 fold of three canocial AH superfamily members showing the conserved metal ions (black) and ligands (white).


AH superfamily members all share metal-assisted polarization of substrate(s) as the common denominator in their catalytic mechanisms.  In the most typical case where hydrolytic cleavage is carried out, the metal catalyzes deprotonation of a water molecule to generate a hydroxide anion which is correspondingly stabilized by coordination to the metal ligand(s).  Subsequent nucleophilic attack by the hydroxide on the substrate results in formation of a tetrahedral (carbon-hetero atom bond) or trigonal, bipyramidal (phosphorus-oxygen bond) intermediate which collapses with concomitant protonation of the leaving group by an Asp residue at the end of the 8th β-strand (Figure AH3).   Although this is the prototypical reaction, the AH superfamily shows extreme functional diversity and a significant variation is illustrated in uronate isomerase family which catalyzes adol-keto conversions of hexuronic acids in carbohydrate metabolism.  Mechanistic and structural studies indicate retooling of active site residues allows for the necessary acid/base catalysis via a cis-endiol intermediate (Figure AH3).  Intriguingly, substrate oxygens distal to the site of chemistry mimic the hydroxide as metal ligands.  This in turn may also activate the distal substrate hydroxyl for intramolecular deprotonation akin to metal activation of water as seen among the more canonical AH members. 


Figure AH3. Representative reaction mechanisms of dihydroorotase and uronate isomerase showing metal-assisted activation of the substrate(s) (top). Representative assigned functions in the AH superfamily highlighting the reactive bond in red (bottom).


Functional assignment in the AH superfamily using the integrated sequence/structure-based strategy first developed during the Program Project effort (P01 GM071790) proved an unparalleled success.  Regardless, the vastness and complexity of the AH superfamily leaves a significant portion uncharacterized and many hurdles to overcome. Among other challenges, of pressing consideration is representation of unknown metabolites in real and in silico libraries.  Extensive collaboration with the Computation and Microbiology Cores aims to address challenges through both exploitation of traditional strategies and development of novel solutions.  As such, the AH superfamily serves as an excellent mature model system to continue development and refinement of the sequence/structure based strategy that is the goal of the EFI. 


Click here to learn about AH target selection during the project perio»  

Click here to view experimental data for AH targets »


Representative References

  • Structural and catalytic diversity within the amidohydrolase superfamily. Seibert CM, Raushel FM.  (2005) Biochemistry 44, 6383-91.
  • Structure-based activity prediction for an enzyme of unknown function. Hermann JC, Marti-Arbona R, Fedorov AA, Fedorov E, Almo SC, Shoichet BK, Raushel FM. (2007) Nature 448, 775-9.
  • The mechanism of the reaction catalyzed by uronate isomerase illustrates how an isomerase may have evolved from a hydrolase within the amidohydrolase superfamily. Nguyen TT, Fedorov AA, Williams L, Fedorov EV, Li Y, Xu C, Almo SC, Raushel FM. (2009) Biochemistry 48, 8879-90.
  • Functional identification and structure determination of two novel prolidases from cog1228 in the amidohydrolase superfamily.  Xiang DF, Patskovsky Y, Xu C, Fedorov AA, Fedorov EV, Sisco AA, Sauder JM, Burley SK, Almo SC, Raushel FM. (2010) Biochemistry 49, 6791-803.