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Mobile Loop Dynamics in Adenosyltransferase Control

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Mobile Loop Dynamics in Adenosyltransferase Control Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B12 Romila Mascarenhasa,1, Markus Ruetza,1, Liam McDevitta, Markos Koutmosb,c, and Ruma Banerjeea,2 aDepartment of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0600; bDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109-0600, and cDepartment of Biophysics, University of Michigan, Ann Arbor, MI 48109-0600 Edited by Amie K. Boal, Pennsylvania State University, State College, PA, and accepted by Editorial Board Member Stephen J. Benkovic October 20, 2020 (received for review April 16, 2020) Cobalamin is a complex organometallic cofactor that is processed Human and Methylobacterium extorquens (Me) ATR, which have and targeted via a network of chaperones to its dependent been characterized most extensively, also double as chaperones, enzymes. AdoCbl (5′-deoxyadenosylcobalamin) is synthesized transferring AdoCbl directly to MCM (Fig. 1A) rather than re- from cob(II)alamin in a reductive adenosylation reaction catalyzed leasing the high-value product into solution (12–17). Cofactor by adenosyltransferase (ATR), which also serves as an escort, de- loading onto MCM is gated by the GTPase activity of the G livering AdoCbl to methylmalonyl-CoA mutase (MCM). The mech- protein chaperone CblA (18). Despite the overall structural and anism by which ATR signals that its cofactor cargo is ready functional conservation of the cofactor loading processes, there (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. are important differences in regulation between the human (19) In this study, we have obtained crystallographic snapshots that – reveal ligand-induced ordering of the N terminus of Mycobacte- and the better-characterized bacterial systems (15 17). However, rium tuberculosis ATR, which organizes a dynamic cobalamin bind- there is virtually no information on the synthesis of AdoCbl by ing site and exerts exquisite control over coordination geometry, Mtb ATR or its delivery to MCM. reactivity, and solvent accessibility. Cob(II)alamin binds with its The PduO class of ATRs is one of three found in nature, in dimethylbenzimidazole tail splayed into a side pocket and its cor- addition to CobA and EutT, that have arisen via convergent rin ring buried. The cosubstrate, ATP, enforces a four-coordinate evolution (20). The PduO ATRs are homotrimers in which the cob(II)alamin geometry, facilitating the unfavorable reduction to active sites reside at subunit interfaces. The human and Me cob(I)alamin. The binding mode for AdoCbl is notably different ATRs bind AdoCbl in the “base-off” state, which specifically BIOCHEMISTRY from that of cob(II)alamin, with the dimethylbenzimidazole tail refers to the conformation in which the endogenous 5,6-dime- tucked under the corrin ring, displacing the N terminus of ATR, thylbenzimidazole (DMB) base is not the α- or lower axial ligand which is disordered. In this solvent-exposed conformation, AdoCbl to cobalt. The “base-on” to base-off switch increases the redox undergoes facile transfer to MCM. The importance of the tail in potential by ∼100 mV (21) and represents a strategy for easing cofactor handover from ATR to MCM is revealed by the failure of 5′-deoxyadenosylcobinamide, lacking the tail, to transfer. In the the challenge of reducing cob(II)alamin to cob(I)alamin, which ′ absence of MCM, ATR induces a sacrificial cobalt–carbon bond ho- subsequently attacks the C5 -carbon of ATP displacing triphos- molysis reaction in an unusual reversal of the heterolytic chemistry phate (PPPi) and forming AdoCbl (Fig. 1A). It is not known that was deployed to make the same bond. The data support an whether a dedicated or shared reductase supports ATR activity. important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites. Significance cobalamin | crystal structure | cofactor | trafficking | kinetics Coenzyme B12 (or 5′-deoxyadenosylcobalamin [AdoCbl]) is a cofactor for methylmalonyl-CoA mutase (MCM), which is im- he persistence of Mycobacterium tuberculosis (Mtb), the portant for propionate metabolism in Mycobacterium tuber- Tcausative pathogen of tuberculosis, is due in part to its culosis and for anaplerosis in humans. AdoCbl is synthesized by metabolic agility in adapting to hostile and nutrient-restricted adenosyltransferase (ATR), which doubles as an escort, deliv- host environments. In particular, the glucose-poor environment ering the cofactor to MCM. The mechanism by which this large of phagosomes promotes mycobacterial subsistence on a diet of cofactor is translocated from ATR to MCM is not known. Our host-derived lipids (1). Catabolism of cholesterol and the crystal structures of M. tuberculosis ATR reveal that mobile β-oxidation of odd-chain fatty acids lead to propionyl-CoA (2), loops dynamically create a customized pocket to control co- which can be utilized via the methylcitrate cycle or the vitamin factor coordination and conformation, regulating its reactivity and transportability. These changes also control solvent ex- B12-dependent methymalonyl-CoA pathway (3). Optimal growth on propionate is dependent on both pathways being posure and signaling to MCM when ATR is ready to transfer operational (4). cargo, in a process where the cofactor tail plays an important role in the handover between proteins. Mtb harbors the requisite set of chaperones to support B12- dependent methylmalonyl-CoA mutase (MCM, Rv1492/1493), Author contributions: R.M., M.R., and R.B. designed research; R.M., M.R., and L.M. per- including adenosyltransferase (ATR or MMAB, Rv1314c) and a formed research; R.M., M.R., L.M., M.K., and R.B. analyzed data; and R.M., M.R., and R.B. GTPase (MMAA or CblA, Rv1496) that have human orthologs wrote the paper. (5–7). MCM isomerizes methylmalonyl-CoA derived via car- The authors declare no competing interest. boxylation of propionyl-CoA, to succinyl-CoA (8, 9), and is This article is a PNAS Direct Submission. A.K.B. is a guest editor invited by the susceptible to inactivation by the immunometabolite, itaconyl- Editorial Board. CoA (10). Mtb does not biosynthesize B12 but scavenges it Published under the PNAS license. 1 from its host (11). Once inside, B12 is assimilated into the bio- R.M. and M.R. contributed equally to this work. logically relevant derivatives, methylcobalamin and 5′- 2To whom correspondence may be addressed. Email: [email protected]. deoxyadenosylcobalamin (AdoCbl). This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ATR is tasked with the synthesis of AdoCbl (Fig. 1A), the doi:10.1073/pnas.2007332117/-/DCSupplemental. cofactor for MCM and type II ribonucleotide reductase in Mtb. www.pnas.org/cgi/doi/10.1073/pnas.2007332117 PNAS Latest Articles | 1of11 Downloaded by guest on September 26, 2021 Fig. 1. Cobalamin binding to and functions of ATR. (A) In the presence of ATP, ATR binds cob(II)alamin in an unfavorable 4-c geometry. Following a one electron reduction, ATR catalyzes the adenosylation of cob(I)alamin to form 5-c base-off AdoCbl (455 nm), which is transferred to the MCM-CblA complex with concomitant hydrolysis of GTP. In the absence of AdoCbl transfer, the newly formed Co-C bond is weakened in the ternary ATR•AdoCbl•PPPi complex leading to a species with an absorption maximum at 440 nm. In the presence of oxygen, Co-C bond cleavage is observed with concomitant formation of hydroperoxyadenosine (Ado-OOH) and cob(II)alamin, with an absorption maximum at 464 nm, which upon reduction, can serve in another cycle of AdoCbl synthesis. Alternatively, further oxidation of cob(II)alamin leads to aquocobalamin, which is released into solution due to its weak affinity for ATR. (B)In- creasing concentrations of ATR were added to cob(II)alamin (50 μM, black) in anaerobic Buffer A at 25 °C in the presence of 5 mM ATP. Spectra were recorded 5 min after each addition (gray lines). Binding of cob(II)alamin to ATR•ATP results in a strong absorption peak at 464 nm (red, final spectrum). (C) The change in absorbance at 464 nm (in B) versus ATR monomer concentration yielded KD = 0.44 ± 0.08 μM (mean ± SD, n = 3). (D) Increasing concentration of ATR were added to AdoCbl (30 μM, black) in Buffer A at 25 °C and spectra were recorded 5 min after each addition (gray lines). ATR binding resulted in a spectral shift from 525 nm to 455 nm (red, final spectrum). (E) The change in absorbance at 525 nm (in D) versus ATR monomer concentration yielded KD = 0.92 ± 0.1 μM (mean ± SD, n = 4). The cobalt-carbon (Co-C) bond in B12 is typically cleaved and ATP involves a chemically expensive SN2 reaction, con- heterolytically in methylcobalamin-dependent methyl transfer suming three high-energy phosphate bonds. Kinetics favor the reactions but homolytically in AdoCbl-dependent isomerase re- direct transfer of the newly synthesized AdoCbl product to MCM actions (22). ATR, in a remarkable display of chemical versa- (Fig. 1A). When this option is unavailable, human ATR reverses tility, catalyzes both types of Co-C bond manipulations in the chemical course and cleaves the Co-C bond homolytically, gen- same active site (19). The synthesis of AdoCbl from cob(I)alamin erating the radical pair, cob(II)alamin and the 5′-deoxyadenosyl 2of11 | www.pnas.org/cgi/doi/10.1073/pnas.2007332117 Mascarenhas et al. Downloaded by guest on September 26, 2021 radical that is quenched by oxygen, forming hydro- peroxyadenosine (Fig. 1A). Under anaerobic conditions, a weakened but intact Co-C bond is seen as evidenced by the formation of a diamagnetic species with absorption maxima at 389 and 439 nm, in contrast to the starting 455 nm, corre- sponding to base-off AdoCbl (19). The presence of PPPi in the ternary product complex is key to facilitating the Co-C bond homolysis step. In its absence, a third route is promoted: That is, loss of AdoCbl into solution, which is augmented by the R186Q patient mutation (19).
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