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

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.

Characterizing metal-binding sites in proteins with X-ray crystallography

“…The analysis shows that the molybdenum content in the sample is below the detection limit of ICP-OES, suggesting the absence of the molybdenum cofactor in the protein. The results are in accordance with current knowledge that the cofactor is produced only under anaerobic conditions 141 . Additionally, the analysis shows Ni 2+ ions are present in a protein sample eluted from a Ni-NTA resin (most likely bound to the His-tag on the N-terminus of the protein) 141 .…”

“…Three different projects are used to illustrate the expected outcomes in steps 1, 2, and 6 (Table 3). We used equine serum albumin (ESA) to illustrate the expected outcome of preliminary analysis in step 1 (PDB ID: 5IJ5) 51 , STM1931 from Salmonella typhimuruim to illustrate thermofluor shift assays (TSA) in step 2 (PDB ID: 4K2H), and a molybdenum cofactor containing chaperone for steroid C25 dehydrogenase from Sterolibacterium denitrificans to illustrate ICP-OES in step 6 141 (Table 3).…”

“… Used proteins include human albumin (HSA) and horse albumin (ESA) (PDB IDs: 5IJF, 5IJ5) 51 , STM1931 from Salmonella typhimuruim (PDB ID: 4K2H), molybdenum cofactor-containing chaperone protein from Sterolibacterium denitrificans 141 , dihydroorotase from Yersinia pestis CO92 (PDB ID: 5V0G). …”

Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms

“…In the genome of S. denitrificans there are seven more genes coding for putative S25DH-like α subunits, five more genes coding for S25DH-like β and γ subunits as well as two copies of specialized chaperone dedicated to Moco insertion (Heider et al 2016 ; Niedzialkowska et al 2017 ). Based on sequence similarity to S25dA, the putative S25DH-like isoenzymes can be divided into three groups: (i) high sequence identity (i.e., S25dA2, 82%; S25dA3, 74%; and S25dA4, 72%), (ii) low sequence identity (S25dA5, S25dA6, S25dA7—app.…”

Crystal structure of thebaine 6-O-demethylase from the morphine biosynthesis pathway

“…Results of preliminary ICP-OES analysis indicated high nickel and only residual iron content. The purification protocol included Ni 2+ affinity chromatography steps that sometimes, as a result of stripping nickel from the resin, may result in sample contamination (Niedzialkowska et al, 2017;Oswald et al, 1997).…”