Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.; Burgos, Emmanuel S.; Gulab, Shivali A.; Schramm, Vern L.

doi:10.1021/bi501487w

Cited by 13 publications

(30 citation statements)

References 34 publications

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“…We explored the substrate specificity of Rv0091 by determining the kinetic constants with MTA, SAH, and 5′-dAdo. 23 These experiments indicate that the preferred substrate for Rv0091 is 5′-dAdo, which displayed a specificity constant ( k cat / K M ) 7-fold greater than MTA and 700-fold greater than SAH (Table 1). MTA was reported as the preferred substrate for M. tuberculosis MTAP, 21 and the addition of phosphate to the Rv0091 reaction mixture did not enhance the rate of MTA hydrolysis.…”

Section: Resultsmentioning

confidence: 87%

“…23 The low catalytic efficiency of enzymes from M. tuberculosis , such as Rv0091, has been attributed to the slow doubling time of M. tuberculosis (24 h) as compared to E. coli (20 min). 24 …”

Section: Resultsmentioning

confidence: 99%

“… a Parameters obtained using a luciferase-based assay 23 at 25 °C. b Adenosine and 2′-deoxyadenosine showed less substrate activity than MTA and were not further characterized. 5′-dAdo, 5′-deoxyadenosine; MTA, 5′-methylthioadenosine; SAH, S -adenosylhomocysteine. …”

Section: Figurementioning

confidence: 99%

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

2016

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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.

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Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

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

2016

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“…, red arrows). As a consequence, the activity of MtnN interfered with AI‐2 synthesis and biofilm formation in a variety of bacterial species (Kang et al ., ; Thomas et al ., ; Han et al ., ). This intermediate step allowed leakage of MTR out of the cell if needed (for MTR transporters identification, see experiments reported in the supplementary text to Borriss et al ., ).…”

Section: General Features Of the Standard Aerobic Mspmentioning

confidence: 99%

Revisiting the methionine salvage pathway and its paralogues

Sekowska

Ashida

Danchin

2018

Microbial Biotechnology

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SummaryMethionine is essential for life. Its chemistry makes it fragile in the presence of oxygen. Aerobic living organisms have selected a salvage pathway (the MSP) that uses dioxygen to regenerate methionine, associated to a ratchet‐like step that prevents methionine back degradation. Here, we describe the variation on this theme, developed across the tree of life. Oxygen appeared long after life had developed on Earth. The canonical MSP evolved from ancestors that used both predecessors of ribulose bisphosphate carboxylase oxygenase (RuBisCO) and methanethiol in intermediate steps. We document how these likely promiscuous pathways were also used to metabolize the omnipresent by‐products of S‐adenosylmethionine radical enzymes as well as the aromatic and isoprene skeleton of quinone electron acceptors.

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“…Catalysis can be affected by local changes in the active site geometry, as well as by alterations in the conformational states of the protein, through allosteric effects and global protein dynamics . Amino acid residues distal from enzyme active sites can contribute to catalysis . However, not all protein motions are coupled to catalysis, and biochemical studies are required to distinguish between slow conformational changes that preorganize the active enzyme scaffold and the fast reorganization during the chemical transformations in the central complexes .…”

Section: Introductionmentioning

confidence: 99%

Contribution of buried distal amino acid residues in horse liver alcohol dehydrogenase to structure and catalysis

Shanmuganatham¹,

Wallace²,

Lee³

et al. 2018

Protein Science

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The dynamics of enzyme catalysis range from the slow time scale (∼ms) for substrate binding and conformational changes to the fast time (∼ps) scale for reorganization of substrates in the chemical step. The contribution of global dynamics to catalysis by alcohol dehydrogenase was tested by substituting five different, conserved amino acid residues that are distal from the active site and located in the hinge region for the conformational change or in hydrophobic clusters. X-ray crystallography shows that the structures for the G173A, V197I, I220 (V, L, or F), V222I, and F322L enzymes complexed with NAD and an analogue of benzyl alcohol are almost identical, except for small perturbations at the sites of substitution. The enzymes have very similar kinetic constants for the oxidation of benzyl alcohol and reduction of benzaldehyde as compared to the wild-type enzyme, and the rates of conformational changes are not altered. Less conservative substitutions of these amino acid residues, such as G173(V, E, K, or R), V197(G, S, or T), I220(G, S, T, or N), and V222(G, S, or T) produced unstable or poorly expressed proteins, indicating that the residues are critical for global stability. The enzyme scaffold accommodates conservative substitutions of distal residues, and there is no evidence that fast, global dynamics significantly affect the rate constants for hydride transfers. In contrast, other studies show that proximal residues significantly participate in catalysis.

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Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

Cited by 13 publications

References 34 publications

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

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

Revisiting the methionine salvage pathway and its paralogues

Contribution of buried distal amino acid residues in horse liver alcohol dehydrogenase to structure and catalysis

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