Nickel quercetinase, a "promiscuous" metalloenzyme: metal incorporation and metal ligand substitution studies.

Dimitrios Nianios,Susanne Fetzner,Sven Thierbach,Lenz Steimer,Dagmar Klostermeier,Pavel Lulchev

doi:10.1186/s12858-015-0039-4

Abstract

BackgroundQuercetinases are metal-dependent dioxygenases of the cupin superfamily. While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni2+ and some other divalent metal ions. This raises the questions of which factors determine metal selection, and which metal ion is physiologically relevant.ResultsMetal occupancies of heterologously produced QueD proteins followed the order Ni > Co > Fe > Mn. Iron, in contrast to the other metals, does not support catalytic activity. QueD isolated from the wild-type Streptomyces sp. strain FLA contained mainly nickel and zinc. In vitro synthesis of QueD in a cell-free transcription-translation system yielded catalytically active protein when Ni2+ was present, and comparison of the circular dichroism spectra of in vitro produced proteins suggested that Ni2+ ions support correct folding. Replacement of individual amino acids of the 3His/1Glu metal binding motif by alanine drastically reduced or abolished quercetinase activity and affected its structural integrity. Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual catalytic activity.ConclusionsHeterologous formation of catalytically active, native QueD holoenzyme requires Ni2+, Co2+ or Mn2+, i.e., metal ions that prefer an octahedral coordination geometry, and an intact 3His/1Glu motif or a 4His environment of the metal. The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance. Ni2+ most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0039-4) contains supplementary material, which is available to authorized users.

Highlights

Quercetinases are metal-dependent dioxygenases of the cupin superfamily
Metal selectivity and catalytic activity of recombinant QueD To enable a systematic study of the metal selectivity of QueD, as well as of the effects of different transition metal ions on quercetinase activity, we produced the enzyme recombinantly in E. coli which was grown in media supplemented with an excess of the metal ion of interest
Because the concentrations of “free” transition metal ions are thought to be in the 10−7 to 10−15 M range [33,34], metal incorporation into QueD in the E. coli expression system may be limited by metal ion availability

Summary

Introduction

While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni2+ and some other divalent metal ions. In contrast to Bacillus QueD, Streptomyces QueD, purified from recombinant E. coli cells grown with various metal ions, was found to be most active with Ni2+, which is highly unusual for oxygenases [3]. In this as well as in other studies on bacterial quercetinases, the proteins were heterologously produced in E. coli. As pointed out in a recent review, when using a highexpression heterologous system for the synthesis of Ni-QueD, “the relevance to metal speciation of protein in the native host remains unclear” [18]

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