Chamond Natalie, Grégoire Christophe, Coatnoan Nicolas, Rougeot Catherine, Freitas-Junior Lucio Holanda, da Silveira José Franco, Degrave Wim M, Minoprio Paola
Department of Immunology, Institut Pasteur, Paris 75724, France.
J Biol Chem. 2003 May 2;278(18):15484-94. doi: 10.1074/jbc.m210830200.
Proline racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 x 10(-4 )and 5.3 x 10(-5) mol of L-proline/s/0.125 microM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline racemases may have therapeutic potential.
脯氨酸消旋酶催化L-脯氨酸和D-脯氨酸对映体的相互转化，迄今为止仅在两个物种中被描述过。最初是在史氏梭菌中发现的，它在活性位点含有半胱氨酸残基，不需要辅因子或其他已知的辅酶。在从致病性人类寄生虫克氏锥虫中分离和克隆出一个基因后，我们最近描述了第一种真核氨基酸（脯氨酸）消旋酶。尽管这种酶在克氏锥虫的复制性非感染形式中位于细胞内，但在寄生虫分化为非分裂感染形式时，会出现膜结合和分泌形式的该酶。脯氨酸消旋酶的分泌形式是一种强大的宿主B细胞有丝分裂原，有助于寄生虫逃避特异性免疫反应。在这里，我们描述了克氏锥虫中的TcPRAC基因编码该酶的功能性细胞内或分泌形式，它们表现出不同的动力学特性，这可能与其相对催化效率有关。尽管酶同工型的Km值处于相似的数量级（29 - 75 mM），但Vmax在2×10⁻⁴至5.3×10⁻⁵摩尔L-脯氨酸/秒/0.125微摩尔同二聚体重组蛋白之间变化。使用酶特异性抑制剂以及通过对活性位点Cys330残基进行定点诱变来消除酶活性的研究，强化了脯氨酸消旋酶作为抗恰加斯病药物开发关键靶点的潜力。最后，我们提出了脯氨酸消旋酶的蛋白质特征，并表明该酶存在于其他几种具有医学和农业意义的致病和非致病细菌基因组中，但在哺乳动物宿主中不存在，这表明抑制脯氨酸消旋酶可能具有治疗潜力。
