X-Ray snapshots of a pyridoxal enzyme: a catalytic mechanism involving concerted [1,5]-hydrogen sigmatropy in methionine \u03b3-lyase

Dan Sato,Tomoo Shiba,Shigeharu Harada,Tomoyoshi Nozaki,Wataru Yamagata,Tsuyoshi Karaki,Takashi Nakazawa

doi:10.1038/s41598-017-05032-6

Abstract

Pyridoxal 5′-phosphate (PLP)-enzymes are essentially involved in amino acid and amine metabolism of a wide variety of organisms. Despite their extensive biochemical studies, there are little evidence and structural data to comprehensively elaborate the catalytic mechanism. We obtained X-ray snapshots of l-methionine γ-lyase from Entamoeba histolytica (EhMGL), a PLP-enzyme catalyzing the γ-elimination reaction of methionine. Here, we suggest a catalytic mechanism of EhMGL by using the X-ray snapshots covering all stages of this multistep catalysis reaction. Initial formation of a Michaelis complex is followed by the migration of double bond from the C4′=Nα–Cα moiety in an intermediate PLP-methionine imine to C4′–Nα=Cα in pyridoxamine 5′-phosphate (PMP)-α,β-dehydromethionine imine without intervention of a putative quinonoid intermediate. The enzyme can facilitate the subsequent γ-elimination of methanethiol by the possible general acid-base catalysis of Tyr108 for the E1cB mechanism, enabling to form the ene-imine C4′–Nα=Cα–Cβ=Cγ structure with the s-cis conformation, which is prerequisite for the non-enzymatic symmetry-allowed suprafacial [1,5]-hydrogen shift to complete the catalytic cycle by releasing α-ketobutyrate. The mechanism based on the X-ray snapshots is consistent with the reactivity of MGL toward methionine analogues. The generality of such a mechanism involving non-enzymatic concerted reaction in other PLP enzymes is discussed.

Highlights

Many enzymes involved in amino acid and amine metabolism bind pyridoxal 5′-phosphate (PLP) as a cofactor and catalyze a wide range of biologically important reactions such as transamination, racemization, deamination, decarboxylation, isomerization, and β- and γ-elimination/substitution1. l-Methionine γ-lyase (MGL: EC 4.4.1.11), a Pyridoxal 5′-phosphate (PLP)-dependent enzyme involved in the transsulfuration pathway, catalyzes the conversion of l-methionine to α-ketobutyric acid through the elimination of methanethiol from l-methionine
Based on the reasonable assumption that these intermediates were produced in the crystals by catalysis of crystalline EhMGL1 from the Michaelis complex, and represent snapshots of the γ-elimination reaction, we propose here the catalytic mechanism for this enzyme (Fig. 3)
We followed the multi-step reactions of a PLP-dependent enzyme, methionine γ-lyase, by matching the steps with the relevant snapshots of intermediate structures elucidated by X-ray crystallography

Summary

Introduction

Many enzymes involved in amino acid and amine metabolism bind pyridoxal 5′-phosphate (PLP) as a cofactor and catalyze a wide range of biologically important reactions such as transamination, racemization, deamination, decarboxylation, isomerization, and β- and γ-elimination/substitution1. l-Methionine γ-lyase (MGL: EC 4.4.1.11), a PLP-dependent enzyme involved in the transsulfuration pathway, catalyzes the conversion of l-methionine to α-ketobutyric acid through the elimination of methanethiol from l-methionine. Many enzymes involved in amino acid and amine metabolism bind pyridoxal 5′-phosphate (PLP) as a cofactor and catalyze a wide range of biologically important reactions such as transamination, racemization, deamination, decarboxylation, isomerization, and β- and γ-elimination/substitution. In protozoan parasites and periodontal bacteria, MGL plays essential roles in energy metabolism, methionine homeostasis, isoleucine biosynthesis, and formation of a sulfide storage molecule, S-methylcysteine. It is essential to obtain a comprehensive set of intermediate structures to describe the reaction pathway catalyzed by MGL. In this manuscript, we suggest a possible catalytic mechanism for MGL from the protozoan parasite Entamoeba histolytica (EhMGL1) based on X-ray snapshots of enzyme-bound reaction intermediates. Non-enzymatic symmetry-allowed suprafacial [1,5]-hydrogen shift at the final stage of the reaction, and this hydrogen shift is supported by a sophisticated symbiosis between PLP and several catalytic residues

Methods

Results

Discussion

Conclusion