The Occurrence of α-Amino-β-phosphonopropionic Acid in the Zoanthid, Zoanthus sociatus, and the Ciliate, Tetrahymena pyriformis<sup>*</sup>

Kittredge, James S.; Hughes, R. R.

doi:10.1021/bi00895a026

Cited by 96 publications

(36 citation statements)

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“…Although it is significantly more active than any of the aminocontaining compounds we tested against native GAT, D-AP3 is a relatively poor substrate compared with the substrates of other N-acetyltransferases (4, 24 -26). Furthermore, although AP3 is a naturally occurring phosphonate, found in Tetrahymena and the soft coral Zoanthus (27)(28)(29), there is no defined physiological role for the D-isomer of AP3.…”

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The Molecular Basis of Glyphosate Resistance by an Optimized Microbial Acetyltransferase

Siehl

Castle

Gorton

et al. 2007

Journal of Biological Chemistry

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GAT is an N-acetyltransferase from Bacillus licheniformis that was optimized by gene shuffling for acetylation of the broad spectrum herbicide, glyphosate, forming the basis of a novel mechanism of glyphosate tolerance in transgenic plants ( Glyphosate (N-phosphonomethylglycine) is a widely used herbicide that acts nonselectively through inhibition of 5-enolpyruvylshikimate-3-phosphate (EPSP) 4 synthase in the aromatic biosynthesis pathway (2). Its efficacy against all plant species, low cost, low mammalian toxicity, and benign environmental impact favor its use in crops that have a transgenic tolerance mechanism (3). The predominant transgene currently in use encodes a bacterial EPSP synthase that is nearly insensitive to inhibition by glyphosate (2). We reported an alternative strategy for glyphosate resistance involving enzymatic conversion of glyphosate to N-acetylglyphosate (NAG) (1). Because the acetylated form of glyphosate has low affinity for the active site of EPSP synthase, it is nonherbicidal. Three variants of an enzyme that catalyzes N-acetylation of the secondary amine of glyphosate were discovered from Bacillus licheniformis. These GAT 5 enzymes are 17 kDa in size, are most active at pH 6.8, and have a K m for acetyl coenzyme A (AcCoA) of 1-2 M. The native enzymes are very poor catalysts for acetylation of glyphosate, with a k cat of 5.3 min Ϫ1 and K m,GPJ of 1.3 mM (for ST401 GAT, referred to hereafter as native GAT). 6 The B. licheniformis enzymes belong to a diverse family of largely uncharacterized bacterial N-acetyltransferases sharing between 30 and 64% amino acid identity to native GAT (Fig. 1). Despite extensive screening of biological amines, including amino acids, nucleotides and antibiotics, the physiological substrates for the native enzymes are unknown (4).To develop gat as a transgene for glyphosate resistance in crops, we subjected the three B. licheniformis enzymes to 11 rounds of gene shuffling and obtained optimized variants with up to a 4,500-fold increase in catalytic efficiency (k cat /K m ) relative to the native enzyme. This was achieved through a combination of improvements in k cat (190-fold) and K m,GPJ (24-fold). When introduced into plants, optimized gat genes confer robust tolerance to glyphosate (1).To gain insight into the molecular mechanism of glyphosate N-acetylation by GAT, we determined the structure of a 7th round GAT variant (termed R7 GAT) in ternary complex with AcCoA and the competitive inhibitor 3-phosphoglycerate (3PG) and in binary complex with oxidized CoA. We also car-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 2JDC, 2JDD) have been deposited in the Protein

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The Molecular Basis of Glyphosate Resistance by an Optimized Microbial Acetyltransferase

Siehl

Castle

Gorton

et al. 2007

Journal of Biological Chemistry

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“…(1) en 1964 et synthttist par Chambers et Isbeil la meme annCe (2). L'intervention de la phosphonoalanine au cours de la biosynthkse de 17AEPA postulee par Kittredge et coll.…”

Section: Introductionunclassified

Synthèse et amination réductrice de phosphonopyruvates: préparation d'acides amino-2 carboxy-2 alkylphosphoniques (β-phosphonoalanine)

Varlet¹,

Collignon²,

Savignac³

1979

Can. J. Chem.

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The reaction of α-copper(I)alkanephosphonates with methyl and ethyl oxalyl chlorides gives methyl or ethyl dialkylphosphonopyruvates which, submitted to reductive amination in ethanol at pH 6–7 with sodium cyanohydridoborate (NaBH3CN) and ammonia, or primary amines, lead to phosphonic amino esters; hydrolysis with diluted HCl gives phosphonic amino acids. These three reactions constitute a general way to β-phosphonoalanine and β-alkyl β-phosphonoalanine.

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“…One of the most widely distributed of such biogenic C-P compounds is 2-amino-3-phosphonopropionic acid, commonly called phosphonoalanine (HOOC-CH(NH 2 )-CH 2 -PO 3 H 2 ); it is formed through the transamination of phosphonopyruvate by many lower organisms, such as the sea anemone Zoanthus sociatus (6) and the protozoan Tetrahymena pyriformis (7). Our earlier studies on its biodegradation showed that 47 of 100 randomly chosen environmental bacterial isolates had the ability to utilize phosphonoalanine as sole phosphorus source for growth (8).…”

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The Purification and Characterization of Phosphonopyruvate Hydrolase, a Novel Carbon-Phosphorus Bond Cleavage Enzyme from Variovorax sp. Pal2

Kulakova

Wisdom

Kulakov³

et al. 2003

Journal of Biological Chemistry

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Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell. The native enzyme had a molecular mass of 63 kDa with a subunit mass of 31.2 kDa. Activity of purified phosphonopyruvate hydrolase was Co 2؉ -dependent and showed a pH optimum of 6.7-7.0. The enzyme had a K m of 0.53 mM for its sole substrate, phosphonopyruvate, and was inhibited by the analogues phosphonoformic acid, 3-phosphonopropionic acid, and hydroxymethylphosphonic acid. The nucleotide sequence of the phosphonopyruvate hydrolase structural gene indicated that it is a member of the phosphoenolpyruvate phosphomutase/isocitrate lyase superfamily with 41% identity at the amino acid level to the carbon-to-phosphorus bond-forming enzyme phosphoenolpyruvate phosphomutase from Tetrahymena pyriformis. Thus its apparently ancient evolutionary origins differ from those of each of the two carbon-phosphorus hydrolases that have been reported previously; phosphonoacetaldehyde hydrolase is a member of the haloacetate dehalogenase family, whereas phosphonoacetate hydrolase belongs to the alkaline phosphatase superfamily of zinc-dependent hydrolases. Phosphonopyruvate hydrolase is likely to be of considerable significance in global phosphorus cycling, because phosphonopyruvate is known to be a key intermediate in the formation of all naturally occurring compounds that contain the carbon-phosphorus bond.

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The Occurrence of α-Amino-β-phosphonopropionic Acid in the Zoanthid, Zoanthus sociatus, and the Ciliate, Tetrahymena pyriformis^*

Cited by 96 publications

References 7 publications

The Molecular Basis of Glyphosate Resistance by an Optimized Microbial Acetyltransferase

The Molecular Basis of Glyphosate Resistance by an Optimized Microbial Acetyltransferase

Synthèse et amination réductrice de phosphonopyruvates: préparation d'acides amino-2 carboxy-2 alkylphosphoniques (β-phosphonoalanine)

The Purification and Characterization of Phosphonopyruvate Hydrolase, a Novel Carbon-Phosphorus Bond Cleavage Enzyme from Variovorax sp. Pal2

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The Occurrence of α-Amino-β-phosphonopropionic Acid in the Zoanthid, Zoanthus sociatus, and the Ciliate, Tetrahymena pyriformis*

Cited by 96 publications

References 7 publications

The Molecular Basis of Glyphosate Resistance by an Optimized Microbial Acetyltransferase

The Molecular Basis of Glyphosate Resistance by an Optimized Microbial Acetyltransferase

Synthèse et amination réductrice de phosphonopyruvates: préparation d'acides amino-2 carboxy-2 alkylphosphoniques (β-phosphonoalanine)

The Purification and Characterization of Phosphonopyruvate Hydrolase, a Novel Carbon-Phosphorus Bond Cleavage Enzyme from Variovorax sp. Pal2

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The Occurrence of α-Amino-β-phosphonopropionic Acid in the Zoanthid, Zoanthus sociatus, and the Ciliate, Tetrahymena pyriformis^*