Kinetic Isotope Effects and Transition State Structure for Human Phenylethanolamine <i>N</i>-Methyltransferase

Stratton, Christopher F.; Poulin, Myles B.; Du, Quan; Schramm, Vern L.

doi:10.1021/acschembio.6b00922

Cited by 13 publications

(18 citation statements)

References 35 publications

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“…Motion along the M40Cα–D141Cα and donor–acceptor coordinates are not correlated (SI Figure S14) and there is little correlation between Δ E and the donor–acceptor distance (Figure 5c). Our DFT calculations show small-negligible charge transfer from the acceptor oxygen to methyl group (Table 2, SI Table S4), which is consistent with previous proposals that MTases possess a loose or dissociative TS (for example, refs (24 and 35)). We cannot rule out a role for donor–acceptor sampling in methyl transfer in COMT, particularly during the proton transfer from catechol to Lys 144, which apparently leads to the formation of a highly preorganized active site with short R (D-A).…”

Section: Resultssupporting

confidence: 90%

“…Sinefungin (adenosyl- l -ornithine), a fungal-derived inhibitor of SAM-dependent MTases, 23 is a nonreactive SAM-analogue with an amine group in place of the transferring methyl group and a CH in place of the sulfur (Figure 1A). As the amine group is likely to be protonated, 24 it should have a more TS-like charge distribution than SAM. Sinefungin derivatives are also known to be good TS analogues of the SAM-dependent lysine methyl transferase, 25 so ternary complexes of COMT containing sinefungin, Mg 2+ and an appropriate catechol should provide a means to probe COMT in a more TS-like conformation.…”

Section: Introductionmentioning

confidence: 99%

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Equatorial Active Site Compaction and Electrostatic Reorganization in Catechol-O-methyltransferase

et al. 2019

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Catechol-O-methyltransferase (COMT) is a model S-adenosyl-l-methionine (SAM) dependent methyl transferase, which catalyzes the methylation of catecholamine neurotransmitters such as dopamine in the primary pathway of neurotransmitter deactivation in animals. Despite extensive study, there is no consensus view of the physical basis of catalysis in COMT. Further progress requires experimental data that directly probes active site geometry, protein dynamics and electrostatics, ideally in a range of positions along the reaction coordinate. Here we establish that sinefungin, a fungal-derived inhibitor of SAM-dependent enzymes that possess transition state-like charge on the transferring group, can be used as a transition state analog of COMT when combined with a catechol. X-ray crystal structures and NMR backbone assignments of the ternary complexes of the soluble form of human COMT containing dinitrocatechol, Mg2+ and SAM or sinefungin were determined. Comparison and further analysis with the aid of density functional theory calculations and molecular dynamics simulations provides evidence for active site “compaction”, which is driven by electrostatic stabilization between the transferring methyl group and “equatorial” active site residues that are orthogonal to the donor–acceptor (pseudo reaction) coordinate. We propose that upon catecholamine binding and subsequent proton transfer to Lys 144, the enzyme becomes geometrically preorganized, with little further movement along the donor–acceptor coordinate required for methyl transfer. Catalysis is then largely facilitated through stabilization of the developing charge on the transferring methyl group via “equatorial” H-bonding and electrostatic interactions orthogonal to the donor–acceptor coordinate.

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Section: Resultssupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Equatorial Active Site Compaction and Electrostatic Reorganization in Catechol-O-methyltransferase

et al. 2019

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“…Finally, the transition state structure of PNMT, the second closest homologue of NNMT, involves even distribution of positive charge along the entire axis of methyl transfer. 41 If NNMT has a transition state structure similar to that of PNMT, analogues like MS2756, which localize positive charge on a single nitrogen atom, do not accurately capture the transient electrostatics of the methyl transfer transition state. While not positively charged, carbon-based linkers avoid the localized positive charge of MS2756 and other amino-bisubstrate inhibitors.…”

Section: • Results and Discussionmentioning

confidence: 99%

High-Affinity Alkynyl Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT)

Policarpo

Decultot²,

May³

et al. 2019

Preprint

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<div> <div> <div> <div> <p>In this work, structure-based rational design led to the development of potent and selective alkynyl bisubstrate inhibitors of NNMT. The reported nicotinamide-SAM conjugate (named <b>NS1</b>) features an alkyne as a key design element that closely mimics the linear, 180° transition state geometry found in the NNMT-catalyzed SAM → NAM (nicotinamide) methyl transfer reaction. NS1 was synthesized as a single enantiomer and diastereomer in 14 steps and found to be a high-affinity, subnanomolar NNMT inhibitor. An X-ray co-crystal structure and structure-activity relationship (SAR) study revealed the unique ability of an alkynyl linker to span the methyl transfer tunnel of NNMT with ideal shape complementarity. The compounds reported in this work represent the most potent and selective NNMT inhibitors reported to date. The rational design principle described herein could potentially be extended to other methyltransferase enzymes. </p> </div> </div> </div> </div>

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“…PNMT is a rate-limiting and essential enzyme that catalyzes the methylation of noradrenaline to adrenaline [ 16 ], and the only N-methyltransferase that can synthesize adrenaline [ 17 ]. Human PNMT is mainly expressed in the adrenal medulla and also present in human lung tissues [ 17 , 18 ]. Previous studies report that the lungs can synthesize adrenaline locally and regulate adrenaline.…”

Section: Discussionmentioning

confidence: 99%

Spatiotemporal Changes in the Gene Expression Spectrum of the β2 Adrenergic Receptor Signaling Pathway in the Lungs of Rhesus Monkeys

Zheng

Cao

et al. 2021

Lung

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Objective β2 adrenergic receptor (ADRB2) agonists mainly participate in regulation of airway function through the ADRB2-G protein-adenylyl cyclase (AC) signaling pathway; however, the key genes associated with this pathway and the spatiotemporal changes in the expression spectrum of some of their subtypes remain unclear, resulting in an insufficient theoretical basis for formulating the dose and method of drug administration for neonates. Methods We performed sampling at different developmental time points in rhesus monkeys, including the embryo stage, neonatal stage, and adolescence. The MiSeq platform was used for sequencing of key genes and some of their subtypes in the ADRB2 signaling pathway in lung tissues, and target gene expression was normalized and calculated according to reads per kilobase million. Results At different lung-developmental stages, we observed expression of phenylethanolamine N-methyltransferase (PNMT), ADRB2, AC, AKAP and EPAC subtypes (except AC8, AKAP4/5), and various phosphodiesterase (PDE) subtypes (PDE3, PDE4, PDE7, and PDE8), with persistently high expression of AC6, PDE4B, and AKAP(1/2/8/9/12/13, and EZR) maintained throughout the lung-developmental process, PNMT, ADRB2, AC(4/6), PDE4B, and AKAP(1/2/8/9/12/13, EZR, and MAP2)were highly expressed at the neonatal stage. Conclusion During normal lung development in rhesus monkeys, key genes associated with ADRB2–G protein–AC signaling and some of their subtypes are almost all expressed at the neonatal stage, suggesting that this signaling pathway plays a role in this developmental stage. Additionally, AC6, PDE4B, and AKAP(1/2/8/9/12/13, and EZR) showed persistently high expression during the entire lung-developmental process, which provides a reference for the development and utilization of key gene subtypes in this pathway.

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Kinetic Isotope Effects and Transition State Structure for Human Phenylethanolamine N-Methyltransferase

Cited by 13 publications

References 35 publications

Equatorial Active Site Compaction and Electrostatic Reorganization in Catechol-O-methyltransferase

Equatorial Active Site Compaction and Electrostatic Reorganization in Catechol-O-methyltransferase

High-Affinity Alkynyl Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT)

Spatiotemporal Changes in the Gene Expression Spectrum of the β2 Adrenergic Receptor Signaling Pathway in the Lungs of Rhesus Monkeys

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