Abstrakt

Aminoacyl proline synthesis coupled with ATP regeneration using pyruvate phosphate dikinase

Shin Suzuki, Kuniki Kino

Aminoacyl prolines (Xaa-Pro) are valuable materials because they exhibit antihypertensive and hypoglycemic activities. We had previously developed a novel method of amide bond formation using the adenylation domain of nonribosomal peptide synthetase that enabled us to synthesize Xaa-Pro enzymatically. However, this method has two significant issues - the supply of ATP for amino acid activation and inhibition of the adenylation reaction caused by pyrophosphate released from the reaction. To address both of these issues, we focused on pyruvate phosphate dikinase (PPDK), which converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, phosphate, and pyruvate. We constructed an ATP regeneration system using AaPPDK, a PPDK from Acetobacter aceti NBRC 14818T, and applied this system to L-Trp-L-Pro synthesis by the adenylation domain of tyrocidine synthetase A (TycA-A). Using this system, L-Trp-L-Pro production from 1 mM ATP increased to 1.83 mM (2.7-fold), and 0.94 mM L-Trp-L-Pro was synthesized from 1 mM AMP. Moreover, optimization of the enzyme concentrations increased L-Trp-L-Pro production supplemented with 1 mM ATP or AMP to 3.18 mM or 2.19 mM, respectively. AaPPDK successfully reduced ATP addition by converting AMP to ATP and increased the reaction rate by removing PPi. Therefore, we established an efficient production method for Xaa-Pro coupled with the ATP regeneration system from AMP employing AaPPDK.