Conformation of the Intermediates in the Reaction Catalyzed by Protoporphyrinogen Oxidase: An In Silico Analysis.

Abigail L Barker,Hamlin Barnes,Franck E Dayan

doi:10.3390/ijms21249495

Abigail L Barker, Hamlin Barnes + Show 1 more

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https://doi.org/10.3390/ijms21249495

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Abstract

Protoporphyrinogen oxidase (PPO) is a critical enzyme across life as the last common step in the synthesis of many metalloporphyrins. The reaction mechanism of PPO was assessed in silico and the unstructured loop near the binding pocket was investigated. The substrate, intermediates, and product were docked in the catalytic domain of PPO using a modified Autodock method, introducing flexibility in the macrocycles. Sixteen PPO protein sequences across phyla were aligned and analyzed with Phyre2 and ProteinPredict to study the unstructured loop from residue 204–210 in the H. sapiens structure. Docking of the substrate, intermediates, and product all resulted in negative binding energies, though the substrate had a lower energy than the others by 40%. The α-H of C10 was found to be 1.4 angstroms closer to FAD than the β-H, explaining previous reports of the reaction occurring on the meso face of the substrate. A lack of homology in sequence or length in the unstructured loop indicates a lack of function for the protein reaction. This docking study supports a reaction mechanism proposed previously whereby all hydride abstractions occur on the C10 of the tetrapyrrole followed by tautomeric rearrangement to prepare the intermediate for the next reaction.

Highlights

The porphyrin pathway plays a central role in the synthesis of many pigments fundamental to sustaining life across the biological realm [1,2,3]
Biosynthesis of porphyrins consists of 7 core enzymes that culminates in the action catalyzed by protoporphyrinogen oxidase (PPO) converting the colorless precursor protoporphyrinogen IX into the fully conjugated, bright red pigment protoporphyrin IX [3]
We aim to better understand the reaction catalyzed by PPO by docking the substrate and product of the reaction along with relevant reaction intermediates, describe the reaction mechanism in a way that unifies previous data while taking into account the spatial limitation of the catalytic domain of PPO as well as information from substrate orientations upstream in the pathway, and to determine if sequence similarity suggests that the unstructured loop has a function in the reaction or substrate binding

Summary

Introduction

The porphyrin pathway plays a central role in the synthesis of many pigments fundamental to sustaining life across the biological realm [1,2,3]. Macrocyclic tetrapyrroles derived for this pathway have a varying degree of conjugation and can be chelated with metal dications such as the alkaline earth metal Mg2+, transition metals such as Fe2+, Co2+, and Ni2+, and group 12 element Zn2+. These metals are coordinated with the pyrrole nitrogens at the center of these rings. PPO itself is of particular interest because mutations of the protein lead to variegate porphyria diseases in humans and in agriculture it is the protein target of the PPO-inhibiting herbicides which are seeing a resurgence of use in recent years [4,5]

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