Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization

https://doi.org/10.1016/j.pep.2017.03.019Get rights and content

Highlights

  • S25DH chaperone D1 has been purified in aerobic and anaerobic conditions.

  • Molybdenum cofactor (Moco) presence in D1 was confirmed by EXAFS and HPLC analysis.

  • D1 protein has been characterized by Thermofluor Shift Assay.

  • Buffer conditions and small molecules that stabilize D1 have been identified.

  • Methods of selection between Moco-free and Moco-loaded protein are presented.

Abstract

Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon's expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.

Introduction

Molybdoenzymes use a wide range of substrates to catalyze diverse redox reactions in bacteria, plants, and animals. Bacterial molybdoenzymes are responsible, for example, for anaerobic respiration, degradation of aromatic and aliphatic hydrocarbons [1] and oxochlorate [2], [3], and metabolism of arsenate [4] and sulfate [5]. In plants, molybdoenzymes take part in nitrate assimilation (nitrate reductases), purine catabolism (xanthine dehydrogenase) [6], hormone synthesis (aldehyde oxidase), and detoxification (sulfite oxidase) [7]. Similarly, in humans, several molybdoenzymes have been identified including xanthine oxidoreductase [8], sulfite and aldehyde oxidase [9], and mitochondrial amidoxime reducing components 1 and 2 (mARC1 and mARC2) [10], [11]. The plethora of substrates capable of being utilized by molybdoenzymes prompted researchers to investigate their potential applications in bioremediation [1], [2], agriculture [12], drug synthesis and medicine [13].

The crucial component of molybdoenzymes is a structure called the molybdenum cofactor (Moco) [14] that forms the active site of the molybdoenzyme. In Moco, a pterin, known as molybdopterin (MPT), serves as a scaffold for the molybdenum atom. Moco must be synthesized de novo in a cell, and the process of Moco synthesis is complex, involving several synthesis pathways and requiring facilitation by multiple proteins [15]. The cofactor moiety isolated from molybdoenzymes is very unstable, and currently, Moco precursors are obtained through chemical synthesis, or isolation and subsequent derivatization of Moco obtained from natural sources [16].

Proteins belonging to the dimethyl sulfoxide (DMSO) reductase family contain the bis-molybdopterin guanine dinucleotide (bis-MGD) type of Moco. In bis-MGD, the molybdenum atom is coordinated through two dithiolene groups of two MGD moieties [17], [18], [19]. In E. coli, the in vivo process of production of bis-MGD cofactor consists of four main steps: a) the conversion of GTP into cyclic pyranopterin monophosphate (cPMP) by S-adenosyl methionine (SAM)-dependent radical enzymes [20] and cyclic pyranopterin monophosphate synthase accessory protein, b) the insertion of sulfur and formation of molybdopterin (MPT) by MPT synthase [21], [22], c) the insertion of molybdenum, which is sequestered as a molybdate oxyanion (MoO42−) [23] into MPT by molybdopterin adenylyltransferase (MogA) [24] and molybdopterin molybdenumtransferase (MoeA) [18] and d) the further modification of Moco to form a dinucleotide derivative of Moco, the MPT-guanine dinucleotide (MGD) cofactor.

The malfunctions of molybdoenzymes due either to mutations in the enzymes or insufficient molybdenum cofactor production can cause various diseases in humans. For example, xanthinuria can be caused by a mutation in the gene coding for xanthine dehydrogenase [25], and molybdenum cofactor deficiency (MoCD) type A is a rare metabolic disease caused by mutations in genes coding for proteins involved in cofactor synthesis. Improper functioning of molybdoenzymes can result in increased levels of metabolites (sulfites) that are toxic if not broken down. MoCDs are linked to brain dysfunction and neurological damage, treatable by daily injections of cofactor precursor, cPMP [26].

Because redox molybdoenzymes such as steroid C25 dehydrogenase (S25DH) [27], ethylbenzene dehydrogenase (EBDH) [1], nitrate reductase (NarGH) [28], and dimethyl sulfoxide (DMSO) reductase [29], require cofactor insertion in the cytoplasm, they can only be transported through the membrane in a folded form [30]. A Moco insertion step into an apo-enzyme is assisted by a chaperone protein. Almost all molybdoenzymes have a private chaperone protein that binds Moco and inserts it into the target apo-enzyme [31]. Subsequently, molybdoenzymes can be transported through membranes through a TAT-pathway that allows for the translocation of fully folded proteins across biological membranes [32]; TAT-pathway substrates possess a characteristic N-terminal amino acid sequence motif that is recognized by a protein from the TAT-pathway system [33]. Our focus in this work revolved around a D1 chaperone protein that is considered responsible for loading a matured form of Moco to the α subunit of S25DH from Sterolibacterium denitrificans (Chol-1ST, DSMZ 13999T) [34] and for transport regulation of folded S25DH enzyme across a membrane via the TAT-pathway [27].

S25DH catalyzes the oxygen-independent hydroxylation of the tertiary C25 carbon atom of the aliphatic side chain of cholesterol and other sterol compounds as well as sterol derivatives (e.g. calciferol) [13], [35] to the respective tertiary alcohol [27]. S25DH is a heterotrimer with αβγ composition that belongs to the DMSO reductase family. The α subunit contains bis-MGD and an iron-sulfur cluster, the β subunit contains four iron sulfur clusters, and the γ subunit contains a heme b [27]. S25DH can be used as a catalyst in the industrial production of calcifediol, which is used in the treatment of vitamin D3 deficiency and rickets [13], [36], or 25-hydroxycholesterol, which is an important regulator of immune function [37], [38]. An efficient overexpression system could also expedite research regarding the reaction mechanism of hydroxylation of tertiary carbon atoms by structural experiments combined with computational studies [93].

E. coli is a prokaryotic model organism that plays an important role in industrial microbiology, biotechnology, and genetic engineering, both in research and application [39]. It is a facultative anaerobe whose metabolism can change in the presence or absence of oxygen. Under aerobic conditions, E. coli uses O2 as a terminal electron acceptor, while it grows anaerobically by fermentation or respiration [40]. When E. coli respires under anaerobic conditions, it can use acceptors such as nitrate, DMSO, trimethylamine-N-oxide (TMAO), or fumarate [41]. The presence of an electron acceptor induces the expression of a specific reductase: for example, TMAO reductase is expressed in the presence of TMAO, and DMSO and fumarate reductases are expressed when sulfoxides or fumarate are present, respectively [42]. On the molecular level, an FNR (fumarate-nitrate reduction) [43] transcription regulator is responsible for the switch between aerobic and anaerobic respiration in E. coli. Under anaerobic conditions, the FNR monomer acquires the [4Fe-4S]2+ cluster, and the protein dimerizes and binds to the promotor regions of target genes, leading to the activation of genes whose products function in anaerobic energy metabolism [44], [45], [46]. The presence of O2 leads to the decomposition of the [4Fe-4S]2+ cluster and FNR dimer by monomer dissociation [47] and subsequent repression of expression of genes involved in anaerobic respiration.

E. coli is an organism widely used in the overproduction of recombinant proteins. One of the main obstacles in S25DH heterologous overexpression may be the lack of an efficient molybdenum cofactor production by E. coli. Although successful overexpression under anaerobic conditions of recombinant molybdoenzyme such as nitrate reductase A (NarGHI) [48] or DmsABC [41], [49], [50] has been reported, it is unclear if the E. coli Moco loading machinery is compatible with recombinant S. denitrificans proteins.

For proteins overexpressed under aerobic conditions, E. coli can be cultured on either rich, minimal, defined, or undefined media [51], [52] depending on the need of further experiments. When recombinant protein production is carried out in E. coli under anaerobic conditions, an electron acceptor is added to the growth medium to allow for anaerobic respiration. Under anaerobic conditions, the presence of nitrate guarantees the overexpression of nitrate reductase [53]. The addition of DMSO or fumarate to the culture medium ensures that E. coli uses these compounds as terminal electron acceptors for growth.

Apart from studies on the biochemistry of molybdoenzymes, chaperone proteins involved in their maturation, such as NarJ [54], DmsD [55], and TorD [56] from E. coli, have been also studied. So far, little is known about the D1 chaperone responsible for S25DH maturation or cofactor loading. Here, we present our efforts to optimize both the production of the Moco in E. coli during the overexpression of the D1 protein and the yield of the D1 chaperone in both Moco-containing and Moco-free forms. The presented protocols were suitable for further analysis of the protein by extended X-ray absorption fine structure (EXAFS), high performance liquid chromatography (HPLC), and thermofluor shift assays.

Section snippets

Genetic construct preparation

Using the StarGate® system (IBA GmbH, Göttingen, Germany), the D1 gene (GeneBank: JQ292999.1) of S25DH was cloned into the pASG-IBA5 and pASG-IBA35 vectors according to the protocol provided by the manufacturer. The initial pEntry vector with the D1 gene was obtained from a laboratory stock (the D1 gene was cloned from S. denitrificans genomic DNA). The resulting plasmids were referred to as pASG-IBA5+D1 and pASG-IBA35 + D1. To prepare expression vectors using the MCSG system [57], the gene

Results

The D1 chaperone is a link between E. coli-synthesized Moco and recombinant overexpressed S25DH. The first goal of this project was to optimize growth conditions for efficient production of the Moco-loaded D1 chaperone which, would allow for protocols suitable for producing active recombinant S25DH. The second goal was to develop a method for obtaining high amounts of soluble protein for biophysical and biochemical studies. The developed methods should allow for selective production of both

Discussion

Optimizing the recombinant expression of complex and multi-subunit enzymes that rely on diverse cofactors is a daunting task. The process of overexpression of such enzymes in an active form is much more complicated. For example, when proteins such as S25DH are assembled and loaded with a redox cofactor within the cytoplasm, they must be transported through the membrane in a fully folded form to the dedicated periplasmic space [72], which involves tight regulations by specific chaperones for

Conclusions

An efficient system for molybdenum cofactor production would positively impact analytical, biochemical, and pharmaceutical studies. We developed efficient methods for production and purification of chaperone proteins containing Moco, which would provide a precursor source of Moco and a high amount of protein for further analysis. The EXAFS and TSA results described here provide background for further studies on Moco cofactor assembly and chaperone functions. The optimized method for anaerobic

Acknowledgements

We thank Przemyslaw J. Porebski and Barat Venkataramany for valuable comments and for critical reading of the manuscript. This research was possible through funding from the Marian Smoluchowski Krakow Research Consortium - a Leading National Research Centre KNOW supported by the Polish Ministry of Science and Higher Education. E.N. was supported by the Foundation for Polish Science. E.N., A.R., and M.S. acknowledge the financial support of the Polish institutions: National Centre for Research

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    Present address: Department of Molecular Physiology and Biological Physics, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA.

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