Heterologous Expression and Site‐Directed Mutagenesis of the 1‐Aminocyclopropane‐1‐Carboxylate Oxidase from Kiwi Fruit

Lay, Venetia J.; Prescott, Andy G.; Thomas, Paul G.; John, P. S.

doi:10.1111/j.1432-1033.1996.0228r.x

Cited by 38 publications

(17 citation statements)

References 22 publications

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“…The existence of this natural and so far unique example provides excellent justification for the corresponding experiment with FHT reported here. While this manuscript was in preparation, results were reported on a site-directed mutagenesis study on ACC oxidase [43], where mutant proteins showed only 1% of the wild-type activity when substitutions were made of His177 and Asp179 to glutamine and glutamic acid, corresponding to the His214 and Asp216 residues of isopenicillin N synthase and to the His220 and Asp222 of FHT.…”

Section: Discussionmentioning

confidence: 99%

Identification of Strictly Conserved Histidine and Arginine Residues as Part of the Active Site in Petunia hybrida Flavanone 3β‐Hydroxylase

Lukačin

Britsch

1997

European Journal of Biochemistry

137

126

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Flavanone 3P-hydroxylase, involved in the biosynthesis of flavonoids, catechins, and anthocyanidins, is a non-heme iron enzyme, dependent on Fez+, molecular oxygen, 2-oxoglutarate, and ascorbate, the typical cofactors of the class of 2-oxoglutarate-dependent dioxygenases.Sequence alignment analysis of various 2-oxoglutarate-dependent dioxygenases and related enzymes revealed eight amino acid residues that seem to be strictly conserved within this group of enzymes. Among these residues, two histidines (His220 and His278) and one aspartic acid (Asp222) were identified as part of the putative iron-binding site and an arginine residue (Arg288) as part of the 2-oxoglutarate binding site, by site-directed mutagenesis and functional analysis of the mutated recombinant enzyme.The mutant genes were expressed in Escherichia coli to give soluble proteins whose molecular masses were in excellent agreement with the wild-type enzyme. Four out of nine mutant enzymes, [Gln78]FHT, [Glnl21]FHT, [Gln264]FHT and [Gln266]FHT, were enzymatically active with activities reduced to 26-57%, implying that the mutated amino acid residues are not essential for catalysis. Replacement of His220 by glutamine and Asp222 by asparagine remarkably reduced the catalytic activity to about 0.15% and 0.4%, respectively. The [Gln220]FHT and [Asn222]FHT enzymes showed a slightly increased K,,, value with respect to iron binding, as compared to the wild-type enzyme. The most drastic effect on the reaction rate of flavanone 3P-hydroxylase was achieved by mutating His278 to glutamine. The mutant had no detectable enzyme activity, indicating that His278 was essential for the catalytic reaction. The observed protection of purified enzyme from inactivation by diethylpyrocarbonate after the addition of cofactors provided further independent confirmation for the involvement of histidine residues in the active site.The substitution of Arg288 by lysine or glutamine induced a precipitous decrease in catalytic activity and a fivefold and 160-fold increase in the Michaelis constants for 2-oxoglutarate, respectively. In addition, the enzymatic activities of the latter two mutant enzymes showed a strong pH dependence in the weakly acidic as well as in the neutral pH range, unlike the wild-type enzyme.These results clearly indicate that Arg288 probably contributes to the specific binding of 2-oxoglutarate at the active site of the enzyme, most likely by providing a positive charge, properly located in order to interact with the S-carboxyl function of 2-oxoglutarate. Furthermore, we conclude that His220, His278 and Asp222 constitute three of the possible ligands for iron binding in the active site of flavanone 38-hydroxylase.

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Section: Discussionmentioning

confidence: 99%

Identification of Strictly Conserved Histidine and Arginine Residues as Part of the Active Site in Petunia hybrida Flavanone 3β‐Hydroxylase

Lukačin

Britsch

1997

European Journal of Biochemistry

137

126

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“…Interestingly, site-directed mutagenesis experiments on ACCO from kiwi fruit shows a great loss of activity when Lys158 is substituted with Ala or Cys (the activity is reduced to less than 1 % of the wild type activity). [38] The model of the ACCO active site includes a bicarbonate anion binding to a protonated lysine cation. Geometry optimizations in gas phase tend to destroy the charge separation and lead to carbonic acid and unprotonated lysine (see Figure 4).…”

Section: Computational Detailsmentioning

confidence: 99%

Ethylene Biosynthesis by 1‐Aminocyclopropane‐1‐Carboxylic Acid Oxidase: A DFT Study

Bassan

Borowski

Schofield

et al. 2006

Chemistry A European J

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The reaction catalyzed by the plant enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) was investigated by using hybrid density functional theory. ACCO belongs to the non-heme iron(II) enzyme superfamily and carries out the bicarbonate-dependent two-electron oxidation of its substrate ACC (1-aminocyclopropane-1-carboxylic acid) concomitant with the reduction of dioxygen and oxidation of a reducing agent probably ascorbate. The reaction gives ethylene, CO(2), cyanide and two water molecules. A model including the mononuclear iron complex with ACC in the first coordination sphere was used to study the details of O-O bond cleavage and cyclopropane ring opening. Calculations imply that this unusual and complex reaction is triggered by a hydrogen atom abstraction step generating a radical on the amino nitrogen of ACC. Subsequently, cyclopropane ring opening followed by O-O bond heterolysis leads to a very reactive iron(IV)-oxo intermediate, which decomposes to ethylene and cyanoformate with very low energy barriers. The reaction is assisted by bicarbonate located in the second coordination sphere of the metal.

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“…The ultimate step of ethylene biosynthesis is the oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) catalyzed by ACC oxidase (ACCO) (3,4), which in vitro requires the addition of Fe(II), O 2 , CO 2 , and ascorbate (5)(6)(7). Previous studies of ACCO invoked a catalytic role for Fe(II) (8)(9)(10), but the nature of this role has remained obscure. The dominant proposal posits the initiating step in catalysis to be ascorbatedriven oxygen activation.…”

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confidence: 99%

Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene

Rocklin

Tierney

Kofman

et al. 1999

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

111

112

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The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O 2 , CO 2 , and ascorbate are required for in vitro enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme⅐(substrate or cofactor)⅐NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O 2 complexes. It is shown here that the ternary Fe(II)ACCO⅐ACC⅐NO complex is readily formed, but no Fe(II)ACCO⅐ascorbate⅐NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with 15 N or 17 O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the ␣-amino and ␣-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O 2 bind to the iron as a step in O 2 activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O 2 , thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.

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Heterologous Expression and Site‐Directed Mutagenesis of the 1‐Aminocyclopropane‐1‐Carboxylate Oxidase from Kiwi Fruit

Cited by 38 publications

References 22 publications

Identification of Strictly Conserved Histidine and Arginine Residues as Part of the Active Site in Petunia hybrida Flavanone 3β‐Hydroxylase

Identification of Strictly Conserved Histidine and Arginine Residues as Part of the Active Site in Petunia hybrida Flavanone 3β‐Hydroxylase

Ethylene Biosynthesis by 1‐Aminocyclopropane‐1‐Carboxylic Acid Oxidase: A DFT Study

Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene

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