Two Structures of Alliinase from Alliium sativum L.: Apo Form and Ternary Complex with Aminoacrylate Reaction Intermediate Covalently Bound to the PLP Cofactor

Shimon, Linda J. W.; Rabinkov, Aharon; Shin, Irina; Miron, Talia; Mirelman, David; Wilchek, Meir; Frolow, Felix

doi:10.1016/j.jmb.2006.11.041

Cited by 59 publications

(60 citation statements)

References 69 publications

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“…The dual use of K88 as a general base for Cα proton abstraction and for stabilization of the resulting PLP-bound carbanion intermediate was also revealed. The second reactive intermediate captured in the dCBS structure, the aminoacrylate species, has been previously observed in the structures of other PLP enzymes (23)(24)(25)(26).…”

Section: Discussionmentioning

confidence: 56%

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

Koutmos

Kabil

Smith

et al. 2010

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

104

247

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The catalytic potential for H 2 S biogenesis and homocysteine clearance converge at the active site of cystathionine β-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes β-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H 2 S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 Å) and an aminoacrylate intermediate (1.55 Å) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory “energy-sensing” CBS domains, named after this protein, suggests a mechanism for allosteric activation by S -adenosylmethionine.

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Section: Discussionmentioning

confidence: 56%

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

Koutmos

Kabil

Smith

et al. 2010

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

104

247

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“…PLP⅐aminoacrylate complex has been observed in five other PLP-dependent enzyme structures determined earlier (41)(42)(43)(44)(45)(46). Occurrence of aminoacrylate at the active site of EcDAPAL could be due to the reduced rate of the catalysis in the crystallization condition (see under "Activity Assay and Spectral Studies").…”

Section: Resultsmentioning

confidence: 75%

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

Bisht

Radha

Bharath

et al. 2012

Journal of Biological Chemistry

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Background: DAPAL is a novel PLP-dependent enzyme involved in the degradation of DAP, a nonstandard amino acid. Results: Striking structural differences were observed between apo-and holo-EcDAPAL, and structure with a PLP-aminoacrylate intermediate bound at the active site was obtained. Conclusion: Enzyme undergoes apo to holo structural transitions and follows a two-base mechanism of catalysis. Significance: The results provide insights into the catalytic mechanism of DAPAL.

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“…Abbreviations: b-S-alliinase, biotinyl-S-alliinase; CD, circular dichroism; DDPM, N-(4-dimethylamino-3,5-dinitrophenyl) maleimide; DTNB (Ellman's reagent), 5,5 0 -dithio-bis-(2-nitrobenzoic acid); DTP, 4,4 0 -dithiodipyridine; DTT, dithiothreitol; EGF, epidermal growth factor; EPR, electron paramagnetic resonance; NTB, nitrothiobenzoate; PBS, phosphate-buffered saline; PLP, pyridoxal-5 0 -phosphate; PLP-AA, PLP-amino acrylate; TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxy.…”

Section: Introductionmentioning

confidence: 99%

Thiol‐disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum)

et al. 2008

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Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5 0 -phosphate-dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a SAS bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored ASH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two ASH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free ASH groups. This allowed the oriented conjugation of alliinase, via the ASH groups, with low-or high-molecular-weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.

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Two Structures of Alliinase from Alliium sativum L.: Apo Form and Ternary Complex with Aminoacrylate Reaction Intermediate Covalently Bound to the PLP Cofactor

Cited by 59 publications

References 69 publications

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

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

Thiol‐disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum)

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