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. 2016 May 16;6(6):603-9.
doi: 10.1002/2211-5463.12071. eCollection 2016 Jun.

Characterization of AmtA, an amidinotransferase involved in the biosynthesis of phaseolotoxins

Mi Li  1 , Li Chen  1 , Zixin Deng  1 , Changming Zhao  1
Affiliations

Characterization of AmtA, an amidinotransferase involved in the biosynthesis of phaseolotoxins

Mi Li et al. FEBS Open Bio. .

Abstract

Phaseolotoxins (PHTs), which are produced by Pseudomonas, belong to a family of phosphoramidate natural products. Two nonproteinogenic amino acid precursors, N(δ)(N'-sulfo-diaminophosphinyl)-ornithine (PSOrn) and homoarginine (hArg), are involved in biosynthesis of PHTs. Amidinotransferase AmtA catalyses the formation of hArg, with arginine and lysine as substrates. AmtA was overexpressed and purified in an Escherichia coli system. An in vitro enzyme assay showed that it has stricter substrate specificity than certain other amidinotransferases. Site-directed mutagenesis experiments showed that the mutation AmtA Met243His244 is an alternative while Met246 is essential for the transamidination activity.

Keywords: amidinotransferase; biosynthesis; homoarginine; phaseolotoxin.

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Figures

Figure 1
Figure 1
Proposed biosynthetic pathway of phaseolotoxins.
Figure 2
Figure 2
Scheme of transamidination reaction. (A) AmtA catalyzed hArg formation; (B) amidine donors tested in this work; (C) amidine acceptors tested in this work.
Figure 3
Figure 3
SDS/PAGE analysis of His‐tagged proteins. (1) Protein molecular weight standards (KDa); (2) His(6)·AmtA; (3) His(6)·AmtA M243P H244N; (4) His(6)·AmtA M246S.
Figure 4
Figure 4
HPLC trace of AmtA‐catalyzed reversible reactions. (A) Forward reaction (with l‐Arg and l‐Lys as substrates); (B) negative control of forward reaction (enzyme AmtA was absent); (C) reverse reaction (with l‐hArg and l‐Orn as substrates); (D) negative control of reverse reaction (enzyme AmtA was absent); (E) negative control (substrates were absent).
Figure 5
Figure 5
HPLC trace of AmtA‐catalyzed transamidination reaction with canavanine as amidine donor. (A) Authorized standard of l‐Arg, one of the targeted reaction products; (B) reaction with canavanine and l‐Orn as substrates; (C) negative control (enzyme AmtA was absent); (D) negative control (substrates were absent).
Figure 6
Figure 6
HPLC trace of AmtA‐catalyzed transamidination reactions with alternative amidine acceptors. (A) With l‐Arg as donor and 6‐Aminocaproic acid as acceptor; (B) with l‐Arg as donor and Norleucine as acceptor; (C) with l‐Arg as donor and l‐Dap as acceptor; (D) with l‐Arg as donor and l‐Gly as acceptor. (i) Authorized standards; (ii) negative control reactions (enzyme AmtA was absent); (iii) transamidination reactions.
Figure 7
Figure 7
HPLC trace of AmtA and its variants catalyzed transamidination reactions. (A) With l‐Arg and l‐Lys as substrates; (B) with l‐Arg and l‐Gly as substrates. (i) Authorized standards; (ii) AmtA‐catalyzed reactions; (iii) AmtA M243P H244N‐catalyzed reactions; (iv) AmtA M246S‐catalyzed reactions.
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