2-Aminoacrylate stress damages diverse PLP-dependent enzymes in vivo

Shen, Wangchen; Borchert, Andrew J.; Downs, Diana M.

doi:10.1016/j.jbc.2022.101970

Cited by 9 publications

(11 citation statements)

References 45 publications

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“…7A ). The presence of a small but detectable level of pyruvate/PLP was consistent with results for some other protein targets of 2AA, which showed a low level of 2AA stress even in the presence of RidA ( 29 ).…”

Section: Resultssupporting

confidence: 85%

“…We considered a scenario in which the IscS Q183P variant restored growth because it was less susceptible to a 2AA attack than wild-type IscS. To assess this possibility, we used an S. enterica system that can monitor 2AA-dependent damage that occurs in vivo ( 15 , 17 , 29 ). PA IscS and PA IscS Q183P proteins were overexpressed and purified from two different S. enterica strains, ridA and wild-type, resulting in four protein samples.…”

Section: Resultsmentioning

confidence: 99%

“…Cofactors were released from each preparation of IscS and IscS Q183P as described previously ( 15 , 17 , 29 ). KOH (30 mM final concentration) was added to 1.5 nmol purified protein in a 100 μL reaction and incubated at room temperature for 10 min.…”

Section: Methodsmentioning

confidence: 99%

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The Cysteine Desulfurase IscS Is a Significant Target of 2-Aminoacrylate Damage in Pseudomonas aeruginosa

Fulton

Irons

Downs

2022

mBio

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

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The Cysteine Desulfurase IscS Is a Significant Target of 2-Aminoacrylate Damage in Pseudomonas aeruginosa

Fulton

Irons

Downs

2022

mBio

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“…Since the protonation of C‐3 of the product is not rate limiting (Table 1 ), why does the enzyme have a strictly conserved histidine residue to perform stereospecific protonation of the product? Enamine intermediates formed by elimination reactions of PLP‐dependent enzymes are known to be toxic to cells because they react as nucleophiles with the internal aldimines of many PLP‐dependent enzymes [ 10 ]. These enzymes are irreversibly inactivated by covalent bond formation [ 11 , 12 , 13 ].…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, this has formed the basis for the design of irreversible inactivators of PLP‐dependent enzymes as potential drugs [ 14 ]. The formation of free aminoacrylates by PLP‐dependent enzymes is well‐documented, for example, in the reaction of threonine deaminase [ 10 , 15 ]. PLP enzymes that catalyze β‐eliminations preferentially release iminopyruvate rather than 2‐aminoacrylate to avoid cytotoxicity [ 16 ].…”

Section: Resultsmentioning

confidence: 99%

His‐163 is a stereospecific proton donor in the mechanism of d‐glucosaminate‐6‐phosphate ammonia‐lyase

2022

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Edited by Christian GriesingerD-Glucosaminate-6-phosphate ammonia-lyase (DGL) catalyzes the conversion of D-glucosaminate-6-phosphate to 2-keto-3-deoxyglutarate-6-phosphate, with stereospecific protonation of C-3 of the product. The crystal structure of DGL showed that His-163 could serve as the proton donor. H163A mutant DGL is fully active in the steady-state reaction, and the pre-steady-state kinetics are very similar to those of wild-type DGL. However, H163A DGL accumulates a transient intermediate with λ max at 293 nm during the reaction that is not seen with wild-type DGL. Furthermore, NMR analysis of the reaction of H163A DGL in D 2 O shows that the product is a mixture of deuterated diastereomers at C-3. These results establish that His-163 is the proton donor in the reaction mechanism of DGL.

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Site‐directed mutagenesis reveals the interplay between stability, structure, and enzymatic activity in RidA from Capra hircus

Rizzi,

Digiovanni,

Degani

et al. 2024

Protein Science

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Reactive intermediate deaminase A (RidA) is a highly conserved enzyme that catalyzes the hydrolysis of 2‐imino acids to the corresponding 2‐keto acids and ammonia. RidA thus prevents the accumulation of such potentially harmful compounds in the cell, as exemplified by its role in the degradation of 2‐aminoacrylate, formed during the metabolism of cysteine and serine, catalyzing the conversion of its stable 2‐iminopyruvate tautomer into pyruvate. Capra hircus (goat) RidA (ChRidA) was the first mammalian RidA to be isolated and described. It has the typical homotrimeric fold of the Rid superfamily, characterized by remarkably high thermal stability, with three active sites located at the interface between adjacent subunits. ChRidA exhibits a broad substrate specificity with a preference for 2‐iminopyruvate and other 2‐imino acids derived from amino acids with non‐polar non‐bulky side chains. Here we report a biophysical and biochemical characterization of eight ChRidA variants obtained by site‐directed mutagenesis to gain insight into the role of specific residues in protein stability and catalytic activity. Each mutant was produced in Escherichia coli cells, purified and characterized in terms of quaternary structure, thermal stability and substrate specificity. The results are rationalized in the context of the high‐resolution structures obtained by x‐ray crystallography.

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2-Aminoacrylate stress damages diverse PLP-dependent enzymes in vivo

Cited by 9 publications

References 45 publications

The Cysteine Desulfurase IscS Is a Significant Target of 2-Aminoacrylate Damage in Pseudomonas aeruginosa

The Cysteine Desulfurase IscS Is a Significant Target of 2-Aminoacrylate Damage in Pseudomonas aeruginosa

His‐163 is a stereospecific proton donor in the mechanism of d‐glucosaminate‐6‐phosphate ammonia‐lyase

Site‐directed mutagenesis reveals the interplay between stability, structure, and enzymatic activity in RidA from Capra hircus

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