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Divergence of structure and function in the haloacid dehalogenase enzyme superfamily: Bacteroides thetaiotaomicron BT2127 is an inorganic pyrophosphatase.

Huang H, Patskovsky Y, Toro R, Farelli JD, Pandya C, Almo SC, Allen KN, Dunaway-Mariano D. (2011) Biochemistry 50, 8937-49. PMCID: PMC3342813

The EFI HAD Bridging Project, Protein Core, and Structure Core combined efforts to determine the function of BT2127, an uncharacterized HAD superfamily member from B. thetaiotaomicron.  The report describes an integrated bioinformatic–structure–mechanism approach which enables reliable tracking and annotation of orthologues.


The explosion of protein sequence information requires that current strategies for function assignment evolve to complement experimental approaches with computationally based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown haloalkanoate dehalogenase superfamily member BT2127 (Uniprot accession code Q8A5 V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics-structure-mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with a k(cat)/K(m) value for pyrophosphate of ~1 × 10(5) M(-1) s(-1)), together with the gene context, support the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure-guided site-directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain-domain association. On the basis of this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172, and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue, β-phosphoglucomutase, control the leaving group size (phosphate vs glucose phosphate) and the position of the Asp acid/base in the open versus closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases.

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Figure 1. Superposition of BT2127 (PDB entry 3QX7) (gray) and L. lactis β-PGM (PDB entry 1O08) (cyan).

Figure 2. Backbone coil depictions of the superposition of the (A) cap-open (royal blue) and cap-closed (cyan) conformations of L. lactis β-PGM (PDB entries 1ZOL and 1O08, respectively) and (B) cap-open (black) and cap-closed (gray) conformations of BT2127 (PDB entries 3QUQ and 3QX7, respectively).

Figure 3. Active site of BT2127 in the cap-closed conformation (PDB entry 3QX7) showing the active site volume calculated in Voidoo (mesh) and the pyrophosphate and β-glucose 1,6-bisphosphate ligands (shown as sticks, phosphorus atoms colored orange) modeled in COOT.

Figure 4. (A) Observed Mg2+ binding site structure of wild-type BT2127 bound to Mg2+ (magenta sphere) (PDB entry 3QUQ). (B) Phosphate binding site observed in the structure of the BT2127 E47N mutant bound with phosphate (phosphorus colored orange) and Ca2+ (green sphere) (PDB entry 3QYP). Water molecules are represented as red spheres, coordination bonds as dashed purple lines, and hydrogen bonds as dashed black lines.

Figure 5. BT2127 active site with pyrophosphate (manually docked). Atom coloring and bonds as in Figure 4.

Figure 6. Phylogenic representation of the biological range of the putative orthologues of BT2127 (blue), β-PGM (brown), and the Archeal pyrophosphatase TON0002 (purple).

Figure 7. Superposition of the structures of BT2127 (gray) (PDB entry 3QX7) and the putative pyrophosphatase from P. horikoshii (teal) (PDB entry 2OM6).

Figure 8. Active site of the putative pyrophosphatase from T. onnurineus BT2127 modeled with pyrophosphate (manually docked) and Mg2+ (derived from the superposition of BT2127 PDB entry 3QUQ). The Mg2+ is shown as a magenta sphere, and the water molecules are represented as red spheres. Atom coloring and bonds as in Figure 4.

2011 HAD Superfamily Publication
Reprinted with permission from Biochemistry.
© 2011 American Chemical Society.