title: Heterologous expression of an active chitin synthase from Rhizopus oryzae.
abstract: Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly.
authors: Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Gto., Mexico.
authors: Holjes
authors: H
authors: Salgado-Lugo
authors: Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Gto., Mexico.
authors: Alejandro
authors: A
authors: Sánchez-Arreguín
authors: Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Gto., Mexico. Electronic address: jruiz@ira.cinvestav.mx.
authors: José
authors: J
authors: Ruiz-Herrera
mesh_terms: D001120:Arginine / Q000737:chemistry; D002686:Chitin / Q000096:biosynthesis* / Q000235:genetics; D002687:Chitin Synthase / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D014508:Urea / Q000737:chemistry
chemical_list: D002686:Chitin; D014508:Urea; D001120:Arginine; D002687:Chitin Synthase
keywords: Chitin synthase; Enzyme expression; Enzyme purification; Inclusion bodies; Rhizopus oryzae