Crystallization of recombinant Bacteroides fragilis glutamine synthetase (GlnN) isolated using a novel and rapid purification protocol

Abstract

a b s t r a c tGlutamine synthetase enzymes (GSs) are large oligomeric enzymes that play a critical role in nitrogen metabolism in all forms of life. To date, no crystal structures exist for the family of large ($1 MDa) type III GS enzymes, which only share 9% sequence identity with the well characterized GSI and GSII enzymes. Here we present a novel protocol for the isolation of untagged Bacteroides fragilis GlnN expressed in an auxotrophic Escherichia coli strain. The rapid and scalable two-step protocol utilized differential precipitation by divalent cations followed by affinity chromatography to produce suitable quantities of homogenous material for structural characterization. Subsequent optimizations to the sample stability and solubility led to the discovery of conditions for the production of the first diffraction quality crystals of a type III GS enzyme.Ó 2010 Elsevier Inc. All rights reserved. IntroductionGlutamine synthetases (GSs) 1 are large oligomeric enzymes that play a central role in nitrogen assimilation, catalysing the condensation of ammonium and glutamate to form glutamine, a precursor for the synthesis of many critical bio-molecules. The ancient [1] and ubiquitous [2] GS superfamily is, therefore, evolutionarily diverse and can be divided into three main groupings, namely GSI, GSII and GSIII, on the basis of amino-acid divergence [3].GSI and GSII enzymes have been purified from numerous bacterial and eukaryotic sources, respectively, over the past five decades of biochemical and structural investigations into their functioning and regulation [4]. The first crystal structure of a GSI enzyme [5] was solved using material prepared from an adenylylation deficient Salmonella typhimurium strain using a combination of simple differential precipitation techniques and nucleotide-analogue affinity chromatography [6]. Since then, advances in recombinant DNA technology and the advent of affinity purification technologies have greatly facilitated the isolation of GS material suitable for crystallization. The first crystal structure of a GSII enzyme was of the Zea mays GS1a protein [7], which was heterologously expressed in Escherichia coli and purified using a combination of anion exchange and affinity chromatography steps [8]. More recently, several structures of GSII enzymes purified with affinity tags [9,10] have been reported.In comparison, GSIII enzymes have only been isolated and characterized from a small number of sources [11][12][13] despite the widespread occurrence of homologues in a number of evolutionarily divergent organisms [14][15][16], including, the anaerobic pathogenic protozoan Trichomonas vaginalis [17]. To date, the only structural information describing the large GSIII enzymes ($1 MDa), which share only $9% sequence identity with the GSI enzymes, is from a low-resolution cryo-EM reconstruction of the GlnN enzyme from the opportunistic human pathogen Bacteroides fragilis [3]. A detailed understanding of the functioning and evolution of these divergent enzymes in light of the known ...

The recA operon: A novel stress response gene cluster in Bacteroides fragilis

“…Glutamine synthetase is an example of such an oxygen-sensitive key catalytic enzyme of anaerobic metabolism. It is responsible for the synthesis of glutamine from glutamate, making it vital to cellular survival [22]. This enzyme relies on an Fe-S cluster at its catalytic centre to facilitate its metabolic function and is thus highly sensitive to oxidative damage [37] making it an ideal target for evaluating the potential of the BCP to act as an antioxidant protein.…”

“…The glnN gene encoding the B. fragilis glutamine synthetase and its product (GSIII) have previously been well characterised [22]. In this study, GSIII activity was further examined by assaying it after exposure to 100 μM H 2 O 2 in the presence and absence of BCP.…”

“…Purification of GSIII was done according to the method of van Rooyen et al [22]. The design of the antioxidant assay was based on the work of Jeong et al [12] with the following modifications: 8 μM of purified BCP protein was used in all experiments; 100 μM H 2 O 2, Trn1 (Sigma), and Trx1 (Sigma) were added at published concentrations [12].…”

Proteolysis of the type III glutamine synthetase fromBacteroides fragiliscauses expedient crystal-packing rearrangements

“…Pure recombinant B. fragilis GSIII was prepared from an Escherichia coli heterologous expression host using a combination of divalent-cation precipitation and affinity chromatography as described previously (van Rooyen et al, 2010).…”

“…Proteolysis was achieved by mixing equal volumes of P. fluorescens CS with pure GSIII (6 mg ml À1 in the final affinity-chromatography elution buffer; van Rooyen et al, 2010) and incubating for 16 h at room temperature. The digested protein was then reconcentrated and buffer-exchanged (three times to one fifth of the volume) in equilibration buffer using a 100 kDa molecular-weight cutoff Nanosep centrifugal concentrator (Pall Corporation) to give a final concentration of 3.5 mg ml À1 .…”