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<p>The most common cyanotoxin microcystin is a cyclic heptapeptide produced by non-ribosomal peptide-polyketide synthetases and tailoring enzymes. The tailoring enzyme McyI, a 2-hydroxyacid dehydrogenase, converts (3-methyl)malate into (3-methyl)oxaloacetate to produce the non-proteinogenic amino acid (3-methyl)aspartate. The reaction is NAD(P)-dependent but the catalytic mechanism remains unclear. Here we describe the crystal structures of McyI at three states: bound with copurified NAD, cocrystallized with NAD/NADP, and cocrystallized with malate or the substrate analogue citrate. An McyI protomer has unusual three nicotinamide cofactor-binding sites, named the NAD-prebound, NADP specific, and non-specific sites. Biochemical studies confirmed the NADP preference during oxidoreductase reaction. Molecular basis for McyI catalysis was revealed by the structures of McyI-NAD binary complex, McyI-NAD-NADP and McyI-NAD-malate ternary complexes, which demonstrate different opening angles between the substrate-binding domain and the nucleotide-binding domain. These findings indicate that McyI is a unique member of the 2-hydroxyacid dehydrogenase superfamily and provide detailed structural insights into its catalytic mechanism. In addition, the structural ensemble representing various binding states offers clues for designing enzyme for bioengineering applications.</p>

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Structural insights into the catalytic mechanism of the microcystin tailoring enzyme McyI

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年4月7日

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  • Xiao Wang,
  • Yue Yin,
  • Wen-Long Cheng,
  • Ya-Fei Duan,
  • Yu-Shuai Li,
  • Jia Wang,
  • Mingzhu Wang,
  • Huai-En Dai,
  • Lin Liu

摘要

The most common cyanotoxin microcystin is a cyclic heptapeptide produced by non-ribosomal peptide-polyketide synthetases and tailoring enzymes. The tailoring enzyme McyI, a 2-hydroxyacid dehydrogenase, converts (3-methyl)malate into (3-methyl)oxaloacetate to produce the non-proteinogenic amino acid (3-methyl)aspartate. The reaction is NAD(P)-dependent but the catalytic mechanism remains unclear. Here we describe the crystal structures of McyI at three states: bound with copurified NAD, cocrystallized with NAD/NADP, and cocrystallized with malate or the substrate analogue citrate. An McyI protomer has unusual three nicotinamide cofactor-binding sites, named the NAD-prebound, NADP specific, and non-specific sites. Biochemical studies confirmed the NADP preference during oxidoreductase reaction. Molecular basis for McyI catalysis was revealed by the structures of McyI-NAD binary complex, McyI-NAD-NADP and McyI-NAD-malate ternary complexes, which demonstrate different opening angles between the substrate-binding domain and the nucleotide-binding domain. These findings indicate that McyI is a unique member of the 2-hydroxyacid dehydrogenase superfamily and provide detailed structural insights into its catalytic mechanism. In addition, the structural ensemble representing various binding states offers clues for designing enzyme for bioengineering applications.