Facile synthesis of 1,1-[2H2]-2-methylaminoethane-1-sulfonic acid as a substrate for taurine α ketoglutarate dioxygenase (TauD)

McCusker, Kevin P.; Klinman, Judith P.

doi:10.1016/j.tetlet.2008.11.063

Cited by 5 publications

(10 citation statements)

References 23 publications

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“…All reagents were purchased from commercial sources and used without further purification unless otherwise indicated. 1,1-[ 1 H 2 ] and 1,1-[ 2 H 2 ]-2-methylaminoethane-1-sulfonic acids were synthesized as previously reported …”

Section: Methodsmentioning

confidence: 99%

An Active-Site Phenylalanine Directs Substrate Binding and C−H Cleavage in the α-Ketoglutarate-Dependent Dioxygenase TauD

McCusker

Klinman

2010

J. Am. Chem. Soc.

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Enzymes that cleave C-H bonds are often found to depend on well-packed hydrophobic cores that influence the distance between the hydrogen donor and acceptor. Residue F159 in taurine alpha-ketoglutarate dioxygenase (TauD) is demonstrated to play an important role in the binding and orientation of its substrate, which undergoes a hydrogen atom transfer to the active site Fe(IV)=O. Mutation of F159 to smaller hydrophobic side chains (L, V, A) leads to substantially reduced rates for substrate binding and for C-H bond cleavage, as well as increased contribution of the chemical step to k(cat) under steady-state turnover conditions. The greater sensitivity of these substrate-dependent processes to mutation at position 159 than observed for the oxygen activation process supports a previous conclusion of modularity of function within the active site of TauD (McCusker, K. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 19791-19795). Extraction of intrinsic deuterium kinetic isotope effects (KIEs) using single turnover transients shows 2- to 4-fold increase in the size of the KIE for F159V in relation to wild-type and F159L. It appears that there is a break in behavior following removal of a single methylene from the side chain of F159L to generate F159V, whereby the protein active site loses its ability to restore the internuclear distance between substrate and Fe(IV)=O that supports optimal hydrogenic wave function overlap.

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Section: Methodsmentioning

confidence: 99%

An Active-Site Phenylalanine Directs Substrate Binding and C−H Cleavage in the α-Ketoglutarate-Dependent Dioxygenase TauD

McCusker

Klinman

2010

J. Am. Chem. Soc.

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“…Succinate concentration was determined using a succinic acid assay kit from Megazyme International Ireland, according to the manufacturer's protocol. 1,1-[ 1 H2] and 1,1-[ 2 H2]-2-methylaminoethane-1-sulfonic acid were synthesized as previously described (33). Stoppedflow assays were performed as in ref.…”

Section: Methodsmentioning

confidence: 99%

Modular behavior of tauD provides insight into the origin of specificity in α-ketoglutarate-dependent nonheme iron oxygenases

McCusker

Klinman

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…To test this hypothesis, we first set out to monitor the fate of the H atoms of UMP by enzymatically synthesizing [1′,3′,4′,5′,5′‐ 2 H]UMP, reacting the purified, labeled substrate with Cpr19 or LipL, and identifying the extent of deuterium retention in the product. Interestingly, di‐deuterated taurine (1‐[ 2 H]taurine) has been used for several mechanistic studies with TauD to reveal a large presteady‐state and steady‐state kinetic isotope effect, consistent with H abstraction by an Fe(IV)‐oxo species . Both 1‐[ 2 H]taurine and U‐[ 2 H]taurine have also been used to define the structural orientation of taurine relative to the iron center .…”

Section: Discussionmentioning

confidence: 99%

Evidence that oxidative dephosphorylation by the nonheme Fe(II), α‐ketoglutarate:UMP oxygenase occurs by stereospecific hydroxylation

et al. 2017

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LipL and Cpr19 are non-heme, mononuclear Fe(II)-dependent, α-ketoglutarate (αKG):UMP oxygenases that catalyze the formation of CO2, succinate, phosphate, and uridine-5′-aldehyde, the last of which is a biosynthetic precursor for several nucleoside antibiotics that inhibit bacterial translocase I (MraY). To better understand the chemistry underlying this unusual oxidative dephosphorylation and establish a mechanistic framework for LipL and Cpr19, we report herein the synthesis of two biochemical probes – [1′,3′,4′,5′,5′-2H]UMP and the phosphonate derivative of UMP – and their activity with both enzymes. The results are consistent with a reaction coordinate that proceeds through the loss of one 2H atom of [1′,3′,4′,5′,5′-2H]UMP and stereospecific hydroxylation geminal to the phosphoester to form a cryptic intermediate, (5′R)-5′-hydroxy-UMP. Thus, these enzyme catalysts can additionally be assigned as UMP hydroxylase-phospholyases.

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Facile synthesis of 1,1-[2H2]-2-methylaminoethane-1-sulfonic acid as a substrate for taurine α ketoglutarate dioxygenase (TauD)

Cited by 5 publications

References 23 publications

An Active-Site Phenylalanine Directs Substrate Binding and C−H Cleavage in the α-Ketoglutarate-Dependent Dioxygenase TauD

An Active-Site Phenylalanine Directs Substrate Binding and C−H Cleavage in the α-Ketoglutarate-Dependent Dioxygenase TauD

Modular behavior of tauD provides insight into the origin of specificity in α-ketoglutarate-dependent nonheme iron oxygenases

Evidence that oxidative dephosphorylation by the nonheme Fe(II), α‐ketoglutarate:UMP oxygenase occurs by stereospecific hydroxylation

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