Quinocarcin and SF-1739, potent antitumor antibiotics, share a common tetracyclic tetrahydroisoquinoline (THIQ)-pyrrolidine core scaffold. Herein, we describe the identification of their biosynthetic gene clusters and biochemical analysis of Qcn18/Cya18 generating the previously unidentified extender unit dehydroarginine, which is a component of the pyrrolidine ring. ATP-inorganic pyrophosphate exchange experiments with five nonribosomal peptide synthetases (NRPSs) enabled us to identify their substrates. On the basis of these data, we propose that a biosynthetic pathway comprising a three-component NRPS/MbtH family protein complex, Qcn16/17/19, plays a key role in the construction of tetracyclic THIQ-pyrrolidine core scaffold involving sequential Pictet-Spengler and intramolecular Mannich reactions. Furthermore, data derived from gene inactivation experiments led us to propose late-modification steps of quinocarcin.
A complex composed of enzyme (horseradish peroxidase, HRP), layered titanate (TiO x ), and magnetic beads (MB) was prepared, and the enzymatic activity of the complex and the repeatable use for the enzyme in the complex were investigated. The HRP/TiO x /MB complex was formed in acetate buffer (pH 4) by electrostatic interaction among the components where HRP and MB have positive charge, whereas TiO x is negatively charged. The resultant complex was sufficiently stable in an aqueous solution, and HRP immobilized on the layered titanate in the complex kept a certain enzymatic activity in which typical enzymatic relationship was observed between substrate concentration and initial reaction rate. By giving a magnetic field using commercial magnets, the HRP/TiO x / MB complex was easily and rapidly recovered, and the recovered complex was able to be used for the next enzymatic reactions. The enzyme/inorganic nanosheet/MB complex proposed in this study is expected to be applied as a new tool in the bioengineering and nanobiotechnological fields.
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