midD-encoded ‘rhizomimosinase’ from Rhizobium sp. strain TAL1145 is a C–N lyase that catabolizes L-mimosine into 3-hydroxy-4-pyridone, pyruvate and ammonia

Abstract

Rhizobium sp. strain TAL1145 catabolizes mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K m, V max and K cat of MidD were 1.27 × 10(-4) mol, 4.96 × 10(-5) mol s(-1) mg(-1), and 2,256.05 s(-1), respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and the addition of 50 μM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 μM PLP restored the catalytic activity of MidD in the reaction containing 50 μM hydroxylamine. The catalytic activity of MidD was found to be specific to mimosine, and the presence of its structural analogs including L-tyrosine, L-tryptophan and L-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C-N lyase.

A Carbon-Nitrogen Lyase from Leucaena leucocephala Catalyzes the First Step of Mimosine Degradation

“…We also expected the mimosine-degrading enzyme from L. leucocephala to be a C-N lyase for two reasons: (1) Smith and Fowden (1966) showed that a C-N lyase from L. leucocephala had mimosine-degrading activity; and (2) recently, we have found that the mimosine degradation geneD (midD) of the L. leucocephala symbiont Rhizobium sp. strain TAL1145 encodes a C-N lyase that degrades mimosine into 3H4P, pyruvate, and ammonia (Negi et al, 2013). Therefore, we analyzed the L. leucocephala iSSH library of 406 cDNA clone sequences to identify any sequence that shows homology to lyases.…”

“…Two of the products of this reaction, pyruvate and ammonia, are the same as the products of mimosine degradation catalyzed by the enzymes from L. leucocephala (Smith and Fowden, 1966) and Rhizobium sp. strain TAL1145 (Negi et al, 2013), in which the third product is either 3,4DHP or its isomer 3H4P. Considering the similarity in the products from the degradation reactions catalyzed by CBL and the mimosine-degrading C-N lyases from L. leucocephala and Rhizobium sp.…”

“…Recently, we have characterized another mimosinedegrading enzyme, rhizomimosinase (Negi et al, 2013), from Rhizobium sp. strain TAL1145, an L. leucocephala root-nodulating bacterial strain.…”

The Chemistry and Biological Activities of Mimosine: A Review

“…Recently, one new knowledge for molecular mechanism of mimosine degradation by midD ‐encoded mimosinase (MidD) is added. The proposed mechanism is based on the high homology and conserved catalytic residues between MidD and cystalysin (Negi et al ., ). The deprotonated amino group of mimosine performs a nucleophilic attack on the C4 position of MidD‐bound pyridoxal‐5′‐phosphate (PLP), which results in the formation of mimosine‐PLP complex.…”

“…Mimosinase was identified as a C―N lyase from seedling extracts of Leucaena and Mimosa pudica , that broke the C―N bond of mimosine to produce 3,4‐DHP (Suda, ; Smith and Fowden, ). Later efforts for biochemical and molecular characterization of mimosinase isolated from various tissues or sources including bacteria and rhizobium were made (Pandey and Dwivedi, ; Negi et al ., ). Mimosinase is suggested to have a homotetrameric structure (Pandey and Dwivedi, ).…”