Divergence of Structure and Function in the Haloacid Dehalogenase Enzyme Superfamily: Bacteroides thetaiotaomicron BT2127 Is an Inorganic Pyrophosphatase

Abstract

The explosion of protein sequence information requires that current strategies for function assignment must 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 # Q8A5V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics/structure/mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with kcat/Km value for pyrophosphate of ∼1 × 105 M−1 s−1), together with the gene context, supports the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of the 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. Based on 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 vs. closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases.

Panoramic view of a superfamily of phosphatases through substrate profiling

“…However, a sampling of k cat /K m values from this analysis ( (20,(22)(23)(24), similar to those of C1 and C2 enzymes (21,22,(25)(26)(27). Overall, the finding that C0 members, which have the most minimal cap inserts, are equally efficient and more specific than the other subfamilies suggests that incorporation of additional domains leads to the expansion of the substrate repertoire in the HADSF.…”

Pyrophosphate-Fueled Na+and H+Transport in Prokaryotes

“…Sequenced genomes containing membrane PPases additionally include genes for soluble PPases, which catalyze energy-dissipating hydrolysis of PP i . Soluble PPase belonging to any of its three unrelated families (57,58) Second, experiments with the photosynthetic bacterium R. rubrum indicated that the H ϩ -PPase gene is expressed throughout all growth phases under anaerobic conditions (60), but its expression level declines progressively in the transition to aerobic conditions in both photosynthetic and fermentative cultures but again increases upon exposure to salt (5,60). Similarly, exposure of plants to anoxia, cold, drought, salt, or nutrient limitation increases the abundance and activity of H ϩ -PPase (61)(62)(63)(64)(65).…”

Prediction of substrate specificity and preliminary kinetic characterization of the hypothetical protein PVX_123945 from Plasmodium vivax

“…HAD superfamily members, in general, are known to act on a wide range of substrates. There are ample examples in literature showing the broad substrate and co-factor specificity of HAD superfamily members (Lu et al, 2005;Nguyen et al, 2008;Huang et al, 2011;Lu et al, 2011). In one study on a HAD superfamily member, BT4131, it was shown that the enzyme can utilize several divalent metal-ion cofactors and different sugar phosphates as substrates with varying kcat and Km values (Lu et al, 2005).…”

“…In yet another paper on HAD phosphohydrolase BT1666, it has been shown that the enzyme possesses broad and overlapping substrate specificity with BT4131 (Lu et al, 2011). A recent paper on understanding the substrate specificity of a HAD superfamily member from Bacteroides thetaiotaomicron (BT2127) resorted to structure determination, substrate screen and site-directed mutagenesis to show that the protein functions as an inorganic pyrophosphatase and also assists in protein synthesis by removal of the product pyrophosphate from tRNA synthase (Huang et al, 2011).…”