Crystal Structure of Escherichia coli Diaminopropionate Ammonia-lyase Reveals Mechanism of Enzyme Activation and Catalysis

Bisht, S.; Radha, Venkatesan; Bharath, S.R.; Kalyani, J.N.; Khan, Farida; Rao, Appaji N; Savithri, H.S.; Murthy, M.R.N.

doi:10.1074/jbc.m112.351809

Cited by 15 publications

(19 citation statements)

References 49 publications

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“…IlvA is not the only enzyme whose mechanism could produce free 2AA in vivo . Organisms have other PLP enzymes whose reaction mechanisms may involve 2AA derived from serine, cysteine, tryptophan, or other metabolites (43–47). PLP-dependent enzymes are ubiquitous, and the need for this cofactor is a selective pressure for the evolution of RidA enzymes to avert damage caused by enamine/imine intermediates.…”

Section: Discussionmentioning

confidence: 99%

RidA Proteins Prevent Metabolic Damage Inflicted by PLP-Dependent Dehydratases in All Domains of Life

Lambrecht

Schmitz

Downs

2013

mBio

128

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Pyridoxal 5′-phosphate (PLP) is a coenzyme synthesized by all forms of life. Relevant to the work reported here is the mechanism of the PLP-dependent threonine/serine dehydratases, which generate reactive enamine/imine intermediates that are converted to keto acids by members of the RidA family of enzymes. The RidA protein of Salmonella enterica serovar Typhimurium LT2 is the founding member of this broadly conserved family of proteins (formerly known as YjgF/YER057c/UK114). RidA proteins were recently shown to be enamine deaminases. Here we demonstrate the damaging potential of enamines in the absence of RidA proteins. Notably, S. enterica strains lacking RidA have decreased activity of the PLP-dependent transaminase B enzyme IlvE, an enzyme involved in branched-chain amino acid biosynthesis. We reconstituted the threonine/serine dehydratase (IlvA)-dependent inhibition of IlvE in vitro, show that the in vitro system reflects the mechanism of RidA function in vivo, and show that IlvE inhibition is prevented by RidA proteins from all domains of life. We conclude that 2-aminoacrylate (2AA) inhibition represents a new type of metabolic damage, and this finding provides an important physiological context for the role of the ubiquitous RidA family of enamine deaminases in preventing damage by 2AA.

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Section: Discussionmentioning

confidence: 99%

RidA Proteins Prevent Metabolic Damage Inflicted by PLP-Dependent Dehydratases in All Domains of Life

Lambrecht

Schmitz

Downs

2013

mBio

128

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“…Among the residues at the active site that showed a change of orientation between eDAPAL and eDAPAL‐DAP complex were Asp120 and Asp189. It was shown previously that D120N and D189N eDAPAL exhibited reduced activity with l ‐ and d ‐DAP . The corresponding residues in sDAPAL are Asp125 and Asp194.…”

Section: Resultsmentioning

confidence: 77%

“…D125E sDAPAL, however, did not exhibit any detectable activity with d ‐DAP, although significant activity was observed with l ‐DAP (Table ). These results suggested that Asp125 might be essential for proton abstraction from d ‐DAP but not l ‐DAP, as observed for eDAPAL . Spectral studies were carried out with both isomers of DAP for D125E sDAPAL and compared with those of sDAPAL as a control.…”

Section: Resultsmentioning

confidence: 87%

“…The results presented suggest that Asp125 is the residue that abstracts the proton from d ‐DAP, whereas Asp194 might be essential for stabilization of the enzyme‐substrate complex. An intramolecular disulfide bond between Cys265 and Cys291, not reported in any other fold‐type II PLP‐dependent enzymes, was observed in the structure of eDAPAL . However, equivalent residues in sDAPAL (Cys271 and Cys299) were not involved in formation of the disulfide bond in solution and their mutation showed that they were not essential for catalysis.…”

Section: Introductionmentioning

confidence: 81%

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Identification of key amino acid residues in the catalytic mechanism of diaminopropionate ammonialyase from Salmonella typhimurium

et al. 2013

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Diaminopropionate ammonialyase (DAPAL), a fold-type II pyridoxal 5′-phosphate-dependent enzyme, catalyzes the a,b-elimination of diaminopropionate (DAP) to pyruvate and ammonia. DAPAL was able to utilize both D-and L-DAP as substrates with almost equal efficiency. Mutational analysis of functionally important residues such as Thr385, Asp125 and Asp194 was carried out to understand the mechanism by which the isomers are hydrolyzed. Further, the putative residues involved in the formation of disulfide bond Cys271 and Cys299 were also mutated. T385S, T385D sDA-PAL were as active with DL-DAP as substrate as sDAPAL, whereas the later exhibited a threefold increase in catalytic efficiency with D-Ser as substrate. Further analysis of these mutants suggested that DAPAL might follow an anti-E 2 mechanism of catalysis that does not involve the formation of a quinonoid intermediate. Of the two mutants of Asp125, D125E showed complete loss of activity with D-DAP as substrate, whereas the reaction with L-DAP was not affected significantly, demonstrating that Asp125 was essential for abstraction of protons from the D-isomer. By contrast, mutational analysis of Asp194 showed that the residue may not be directly involved in proton abstraction from L-DAP. sDAPAL does not form a disulfide bond in solution, although the position of Cys299 and Cys271 in the modeled structure of sDAPAL favored the formation of a disulfide bond. Further, unlike eDAPAL, sDAPAL could be activated by monovalent cations. Mutation of the cysteine residues showed that Cys271 may be involved in coordinating the monovalent cation, as observed in the case of other fold-type II enzymes.

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“…4C). Such an environment around the nitrogen atom of the pyridine ring would not favor its electron withdrawing potential necessary for its co-factor function (32). It is likely that a similar situation is found in the C. albicans THI5p homolog structure (12).…”

Section: Table 2 Quantification Of Iron By the Ferrozine Assaymentioning

confidence: 95%

The Last Piece in the Vitamin B1 Biosynthesis Puzzle

Coquille

Roux

Fitzpatrick

et al. 2012

Journal of Biological Chemistry

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Background:The enzyme synthesizing the pyrimidine moiety (HMP-P synthase or THI5p) of vitamin B 1 is poorly characterized. Results:We determined an atomic model of THI5p and have identified its active site using mutagenesis experiments. Conclusion: THI5p forms a dimer and uses PLP as a substrate rather than as a cofactor. Significance: Our study provides seminal information on the enzymatic mechanism of HMP-P synthase.

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Crystal Structure of Escherichia coli Diaminopropionate Ammonia-lyase Reveals Mechanism of Enzyme Activation and Catalysis

Cited by 15 publications

References 49 publications

RidA Proteins Prevent Metabolic Damage Inflicted by PLP-Dependent Dehydratases in All Domains of Life

RidA Proteins Prevent Metabolic Damage Inflicted by PLP-Dependent Dehydratases in All Domains of Life

Identification of key amino acid residues in the catalytic mechanism of diaminopropionate ammonialyase from Salmonella typhimurium

The Last Piece in the Vitamin B1 Biosynthesis Puzzle

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