Transition State Structure and Inhibition of Rv0091, a 5′-Deoxyadenosine/5′-methylthioadenosine Nucleosidase from Mycobacterium tuberculosis

Abstract

5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme that catalyzes the hydrolysis of the N-ribosidic bond in 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). MTAN activity has been linked to quorum sensing pathways, polyamine biosynthesis, and adenine salvage. Previously, the coding sequence of Rv0091 was annotated as a putative MTAN in Mycobacterium tuberculosis. Rv0091 was expressed in Escherichia coli, purified to homogeneity, and shown to be a homodimer, consistent with MTANs from other microorganisms. Substrate specificity for Rv0091 gave a preference for 5′-deoxyadenosine relative to MTA or SAH. Intrinsic kinetic isotope effects (KIEs) for the hydrolysis of [1′-3H], [1′-14C], [5′-3H2], [9-15N], and [7-15N]MTA were determined to be 1.207, 1.038, 0.998, 1.021, and 0.998, respectively. A model for the transition state structure of Rv0091 was determined by matching KIE values predicted via quantum chemical calculations to the intrinsic KIEs. The transition state shows a substantial loss of C1′–N9 bond order, well-developed oxocarbenium character of the ribosyl ring, and weak participation of the water nucleophile. Electrostatic potential surface maps for the Rv0091 transition state structure show similarity to DADMe-immucillin transition state analogues. DADMe-immucillin transition state analogues showed strong inhibition of Rv0091, with the most potent inhibitor (5′-hexylthio-DADMe-immucillinA) displaying a Ki value of 87 pM.

Transition State Analogue Inhibitors of 5′-Deoxyadenosine/5′-Methylthioadenosine Nucleosidase from Mycobacterium tuberculosis

“…4 The 5′-alkylthio-DADMe-Immucillin inhibitors resemble the Rv0091 transition state structure while incorporating features of the MTA substrate. MT-DADMe-ImmA ( 11 ) incorporates the 5′-methylthio group of MTA and exhibits an inhibition constant of 1.5 ± 0.4 nM (Figure 2 and Table 1).…”

“…Increased hydrophobicity induced by modifying the 5′-substituent group to a 5′-hexylthio ( 14 ) improved the dissociation constant to 87 ± 12 pM. 4 …”

“…25 The K M for 5′-dAdo is 10.9 μ M, and [E], [I], and [S] are the enzyme, inhibitor, and substrate concentrations, respectively. 4 …”

“…Mycobacterium tuberculosis Rv0091 was originally annotated as a MTAN; however, recent studies demonstrate that it functions primarily as a 5′-dAdo nucleosidase. 4 5′-dAdo is generated together with methionine as products of radical SAM-dependent enzyme reactions (Scheme 1). 5 The M. tuberculosis genome encodes 21 open reading frames annotated as radical SAM enzymes based on the sequence analysis identifying an iron–sulfur CX 3 CX 2 C motif that has been linked to radical SAM function.…”

“…4 The DADMe-Immucillins contain a 5′-thio group to mimic the MTAN substrate MTA. A family of the DADMe-Immucillins inhibited Rv0091 with dissociation constants with picomolar to nanomolar affinity.…”