Cloning, expression, and in silico structural modeling of cholesterol oxidase of Acinetobacter sp. strain RAMD in E. coli

Mahmoud, Hoda; El‐Far, Shaymaa W.; Embaby, Amira M.

doi:10.1002/2211-5463.13254

Cited by 11 publications

(9 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The literature suggests that cholestenone can be produced by a limited number of microbes that infect humans including Mycobacteria , Rhodococcus, and Nocardia ( 54 ). In addition, we performed a literature review on the top bacterial causes of pulmonary infections and only identified reports of cholesterol oxidase activity by ChoD orthologs in Acinetobacter ( 55 ), Pseudomonas ( 56 – 58 ), and Serratia ( 59 ). However, there are several studies showing that putative cholesterol oxidases of Pseudomonas produce hydroperoxycholestenone (HCEO) rather than cholestenone ( 56 – 58 ).…”

Section: Discussionmentioning

confidence: 99%

Macrophage global metabolomics identifies cholestenone as host/pathogen cometabolite present in human Mycobacterium tuberculosis infection

Chandra¹,

Coullon²,

Agarwal³

et al. 2022

Journal of Clinical Investigation

View full text Add to dashboard Cite

Mycobacterium tuberculosis ( M. tuberculosis ) causes an enormous burden of disease worldwide. As a central aspect of its pathogenesis, M. tuberculosis grows in macrophages, and host and microbe influence each other’s metabolism. To define the metabolic impact of M. tuberculosis infection, we performed global metabolic profiling of M. tuberculosis –infected macrophages. M. tuberculosis induced metabolic hallmarks of inflammatory macrophages and a prominent signature of cholesterol metabolism. We found that infected macrophages accumulate cholestenone, a mycobacterial-derived, oxidized derivative of cholesterol. We demonstrated that the accumulation of cholestenone in infected macrophages depended on the M. tuberculosis enzyme 3 β -hydroxysteroid dehydrogenase (3 β -Hsd) and correlated with pathogen burden. Because cholestenone is not a substantial human metabolite, we hypothesized it might be diagnostic of M. tuberculosis infection in clinical samples. Indeed, in 2 geographically distinct cohorts, sputum cholestenone levels distinguished subjects with tuberculosis (TB) from TB-negative controls who presented with TB-like symptoms. We also found country-specific detection of cholestenone in plasma samples from M. tuberculosis –infected subjects. While cholestenone was previously thought to be an intermediate required for cholesterol degradation by M. tuberculosis , we found that M. tuberculosis can utilize cholesterol for growth without making cholestenone. Thus, the accumulation of cholestenone in clinical samples suggests it has an alternative role in pathogenesis and could be a clinically useful biomarker of TB infection.

show abstract

Section: Discussionmentioning

confidence: 99%

Macrophage global metabolomics identifies cholestenone as host/pathogen cometabolite present in human Mycobacterium tuberculosis infection

Chandra¹,

Coullon²,

Agarwal³

et al. 2022

Journal of Clinical Investigation

View full text Add to dashboard Cite

show abstract

“…After incubation, the induced cells were harvested by centrifugation at 6,000×g for 20 min at 4 °C and resuspended in 50 mM Tris–HCl buffer, pH 8.0. A previously described technique (Abady et al 2021 ; Mahmoud et al 2021 ) was applied to break down the induced cells. Concisely, the cell pellets were suspended in 4 mL of disruption buffer (50 mM Tris/HCl, pH 7.6; 50 mg/mL lysozyme, and 300 mM NaCl).…”

Section: Methodsmentioning

confidence: 99%

“…Purification of the recombinant expressed EstRag was carried out using a procedure that has been previously described with minor modifications (Mahmoud et al 2021 ). In brief, the resultant soluble portion of cell lysate containing 100 mg of crude protein was loaded onto a 2 mL Ni 2+ -NTA affinity matrix.…”

Section: Methodsmentioning

confidence: 99%

Molecular study on recombinant cold-adapted, detergent- and alkali stable esterase (EstRag) from Lysinibacillus sp.: a member of family VI

Matrawy

Khalil

Embaby

2022

World J Microbiol Biotechnol

Self Cite

View full text Add to dashboard Cite

Cold-adapted esterases have potential industrial applications. To fulfil the global continuous demand for these enzymes, a cold-adapted esterase member of family VI from Lysinibacillus sp. YS11 was cloned on pET-28b (+) vector and expressed in E. coli BL21(DE3) Rosetta cells for the first time. The open reading frame (654 bp: GenBank MT120818.1) encodes a polypeptide (designated EstRag: 217 amino acid residues). EstRag amino acid sequence has conserved esterase signature motifs: pentapeptide (GFSQG) and catalytic triad Ser110-Asp163-His194. EstRag 3D predicted model, built with LOMETS3 program, showed closest structural similarity to PDB 1AUO_A (esterase: Pseudomonas fluorescens); TM-align score program inferences. Purified EstRag to 9.28-fold, using Ni2+affinity agarose matrix, showed a single protein band (25 kDa) on SDS-PAGE, Km (0.031 mM) and Kcat/Km (657.7 s−1 mM−1) on p-NP-C2. Temperature and pH optima of EstRag were 35 °C and 8.0, respectively. EstRag was fully stable at 5–30 °C for 120 min and at pH(s) 8.0–10.0 after 24 h. EstRag activity (391.46 ± 0.009%) was impressively enhanced after 30 min preincubation with 5 mM Cu2+. EstRag retained full stability after 30 min pre-incubation with 0.1%(v/v) SDS, Triton X-100, and Tween-80. EstRag promising characteristics motivate performing guided evolution and industrial applications prospective studies.

show abstract

“…After incubation at 37℃ for 16 h, harvesting of the induced cells was performed by centrifugation at 5, 000 ×g for 5 min. Cell breakage by sonication (Fisher Brand TM Sound Enclosure, Thermo Fisher Scientific Co., USA) was performed according to a previously reported procedure (Mahmoud et al 2021 ; Abady et al 2022 ). The soluble supernatant of the cell lysate was maintained at -20 °C until further processing.…”

Section: Methodsmentioning

confidence: 99%

Recombinant acetylxylan esterase of Halalkalibacterium halodurans NAH-Egypt: molecular and biochemical study

Embaby

Mahmoud²

2022

AMB Expr

Self Cite

View full text Add to dashboard Cite

Acetylxylan esterase plays a crucial role in xylan hydrolysis as the acetyl side-groups restrict endoxylanase action by stearic hindrance. In this study, an acetylxylan esterase (AXE-HAS10: 960 bp & 319 a.a) putative ORF from Halalkalibacterium halodurans NAH-Egypt was extensively studied through heterologous overexpression in Escherichia coli, biochemical characterization, and structural modeling. The AXE-HAS10 tertiary structure was predicted by the Local Meta Threading Server. AXE-HAS10 belongs to the carbohydrate esterase Family 7. Purified to homogeneity AXE-HAS10 showed specific activity (36.99 U/mg), fold purification (11.42), and molecular mass (41.39 kDa). AXE-HAS10 showed optimal pH (8.5) and temperature (40 oC). After 15 h of incubation at pH 7.0–9.0, AXE-HAS10 maintained 100% activity. After 120 min at 35 and 40 oC, the retained activity was 80 and 50%, respectively. At 10 mM Mn2+, Fe3+, K+, and Ca2+ after 30 min, retained activity was 329 ± 15, 212 ± 5.2, 123 ± 1.4, and 120 ± 3.0%, respectively. After 30 min of preincubation with triton x-100, SDS, and CTAB at 0.1% (v/v), the retained activity was 150 ± 19, 88 ± 4, and 82 ± 7%, respectively. At 6.0 M NaCl after 30 min, retained activity was 58%. A 1.44-fold enhancement of beechwood xylan hydrolysis was achieved by AXE-HAS10 and Penicillium chrysogenum DSM105774 β-xylanase concurrently. Present data underpins AXE-HAS10 as a promising AXE for industrial exploitation.

show abstract

Cloning, expression, and in silico structural modeling of cholesterol oxidase of Acinetobacter sp. strain RAMD in E. coli

Abstract: Cholesterol oxidases (CHOXs) are flavin-adenine dinucleotide-dependent oxidoreductases with a range of biotechnological applications. There remains an urgent need to identify novel CHOX family

Cited by 11 publications

References 50 publications

Macrophage global metabolomics identifies cholestenone as host/pathogen cometabolite present in human Mycobacterium tuberculosis infection

Macrophage global metabolomics identifies cholestenone as host/pathogen cometabolite present in human Mycobacterium tuberculosis infection

Molecular study on recombinant cold-adapted, detergent- and alkali stable esterase (EstRag) from Lysinibacillus sp.: a member of family VI

Recombinant acetylxylan esterase of Halalkalibacterium halodurans NAH-Egypt: molecular and biochemical study

Contact Info

Product

Resources

About