Protein-pyridinol thioester precursor for biosynthesis of the organometallic acyl-iron ligand in [Fe]-hydrogenase cofactor.

Takashi Fujishiro,Ulrich Ermler,Seigo Shima,Jörg Kahnt

doi:10.1038/ncomms7895

Abstract

The iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase contains a prominent iron centre with an acyl-Fe bond and is the only acyl-organometallic iron compound found in nature. Here, we identify the functions of HcgE and HcgF, involved in the biosynthesis of the FeGP cofactor using structure-to-function strategy. Analysis of the HcgE and HcgF crystal structures with and without bound substrates suggest that HcgE catalyses the adenylylation of the carboxy group of guanylylpyridinol (GP) to afford AMP-GP, and subsequently HcgF catalyses the transesterification of AMP-GP to afford a Cys (HcgF)-S-GP thioester. Both enzymatic reactions are confirmed by in vitro assays. The structural data also offer plausible catalytic mechanisms. This strategy of thioester activation corresponds to that used for ubiquitin activation, a key event in the regulation of multiple cellular processes. It further implicates a nucleophilic attack onto the acyl carbon presumably via an electron-rich Fe(0)- or Fe(I)-carbonyl complex in the Fe-acyl formation.

Highlights

The iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase contains a prominent iron centre with an acyl-Fe bond and is the only acyl-organometallic iron compound found in nature
Recent findings using structure-to-function strategy[17,18] indicated that the formation of 6-carboxymethyl-guanylylpyridinol from GTP and a pyridinol compound are catalysed by HcgB, which strongly suggested that GP is an intermediate in the Fe centre of the ironguanylylpyridinol (FeGP) biosynthetic pathway and, that its carboxy group is converted to the acyl-iron ligand in subsequent enzymatic reactions[19]
We find that HcgE and HcgF catalyse the formation of a thioester-activated acyl-group using a two-state mechanism reminiscent of that of ubiquitin activation[20]

Summary

Introduction

The iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase contains a prominent iron centre with an acyl-Fe bond and is the only acyl-organometallic iron compound found in nature. Analysis of the HcgE and HcgF crystal structures with and without bound substrates suggest that HcgE catalyses the adenylylation of the carboxy group of guanylylpyridinol (GP) to afford AMP-GP, and subsequently HcgF catalyses the transesterification of AMP-GP to afford a Cys (HcgF)-S-GP thioester Both enzymatic reactions are confirmed by in vitro assays. The structural data offer plausible catalytic mechanisms This strategy of thioester activation corresponds to that used for ubiquitin activation, a key event in the regulation of multiple cellular processes. Recent findings using structure-to-function strategy[17,18] indicated that the formation of 6-carboxymethyl-guanylylpyridinol from GTP and a pyridinol compound are catalysed by HcgB, which strongly suggested that GP is an intermediate in the FeGP biosynthetic pathway and, that its carboxy group is converted to the acyl-iron ligand in subsequent enzymatic reactions[19]. On the basis of the findings, possible Fe-acyl formation mechanisms are proposed

Methods

Results

Conclusion