Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism

Abstract

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, this data provides important new insights to the factors determining stability, substrate recognition and the structural mechanism of hydrolysis in this industrially important group of enzymes.

Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA

“…Given that this variant shows a reduced stereospecificity in hydrolysis of phytate it is possible that the enzyme may be able to bind the substrate analogue in multiple orientations. This phenomenon was previously observed for the MINPP phytase from B. longum [ 40 ]. Consequently, the sulfomonoester groups at positions 1D-3 and 1D-6 of the inositol ring of InsS 6 were simultaneously modelled in the primary specificity pocket adjacent to His17 to model static disorder in the binding of the inhibitor ( Figure S4 ).…”

“…Indeed, the substrate binds so as to insert the 1D-3-phosphate in the catalytic pocket despite the pronounced 1D-6-phytase activity of the enzyme. In many cases, myo -inositol hexakissulfate (InsS 6 ) is reported to act as a competitive inhibitor of phytases and in crystal structures is assumed to mimic the substrate by adopting a pseudo-productive binding mode [ 25 , 40 , 44 , 45 ]. Attempts were therefore made to solve the crystal structure of InsS 6 -inhibited AppA by soaking crystals grown at pH 6.5; however, no resolvable electron density for the bound inhibitor was observed.…”

“…This trend is broadly characteristic of all HP2 phytases for which crystal structures are available. However, we note that specificity subsites D and E are more highly populated in the MINPP from Bifidobacterium longum where residues of an unusual polypeptide insertion in the α-domain contribute to additional interactions with the substrate [ 40 ].…”

“…At residue position 305, the T305E substitution found in the AppA-HDE and -HAE variants results in sidechain χ 1 angles of 46° and 65°, respectively, for the glutamate residue. The residue therefore adopts the g − conformation rather than the preferred g + conformation for glutamate observed in α-helical environments [ 49 , 50 , 51 ], which is seen in bacterial MINPPs for which crystal structures are available [ 25 , 40 ]. The g + rotamer is required for the carboxyl group of this residue to approach the phosphomonoester bridging oxygen in the specificity pocket A to allow it to function as a proton donor in catalysis.…”

“…MINPPs were so-named because of their broad substrate specificity compared with other HP2Ps. However, unlike other HP2P enzymes, MINPPs lack a strong stereospecificity towards phytic acid, producing instead a variety of InsP 5 s and lower inositol polyphosphates [ 25 , 37 , 38 , 39 , 40 ]. This led to the suggestion that MINPPs may provide a route to efficient dephosphorylation of dietary phytate which avoids the accumulation of lower inositol polyphosphates observed with animal feed enzymes based on AppA [ 40 ].…”