Structures of purine nucleoside phosphorylase from Mycobacterium tuberculosis in complexes with immucillin-H and its pieces

A structural genomics comparison of purine nucleoside phosphorylases (PNPs) indicated that the enzyme encoded by Mycobacterium tuberculosis (TB-PNP) r esembles the mammalian trimeric structure rather than the bacterial hexamer ic PNPs. The crystal structure of M. tuberculosis PNP in complex with the t ransition-state analogue immucillin-H (ImmH) and inorganic phosphate was so lved at 1.75 Angstrom resolution and confirms the trimeric structure. Bindi ng of the inhibitor occurs independently at the three catalytic sites, unli ke mammalian PNPs which demonstrate negative cooperativity in ImmH binding. Reduced subunit interface contacts for TB-PNP, compared to the mammalian e nzymes, correlate with the loss of the cooperative inhibitor binding. Mamma lian and TB-PNPs both exhibit slow-onset inhibition and picomolar dissociat ion constants for ImmH, The structure supports a catalytic mechanism of rea ctant destabilization by neighboring group electrostatic interactions, tran sition-state stabilization, and leaving group activation. Despite an overal l amino acid sequence identity of 33%, between bovine and TB-PNPs and almos t complete conservation in active site residues, one catalytic site differe nce suggests a strategy for the design of transition-state analogues with s pecificity for TB-PNP. The structure of TB-PNP was also solved to 2.0 Angst rom with 9-deazahypoxanthine (9dHX), iminoribitol (IR), and PO4 to reconstr uct the ImmH complex with its separate components. One subunit of the trime r has 9dHX, IR, and PO, bound, while the remaining two subunits contain onl y 9dHX. In the filled subunit, 9dHX retains the contacts found in the ImmH complex. However, the region of IR that corresponds to the oxocarbenium ion is translocated in the direction of the reaction coordinate, and the nucle ophilic phosphate rotates away from the IR group. Loose packing of the piec es of ImmH in the catalytic site establishes that covalent connectivity in ImmH is required to achieve the tightly bound complex.