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Kusada H, Tamaki H. Evidence for the Worldwide Distribution of a Bile Salt Hydrolase Gene in Enterococcus faecium Through Horizontal Gene Transfer. Int J Mol Sci 2025; 26:612. [PMID: 39859326 PMCID: PMC11765501 DOI: 10.3390/ijms26020612] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2024] [Revised: 01/04/2025] [Accepted: 01/06/2025] [Indexed: 01/27/2025] Open
Abstract
Bile salt hydrolase (BSH), a probiotic-related enzyme with cholesterol-assimilating and anti-hypercholesterolemic abilities, has been isolated from intestinal bacteria; however, BSH activity of bacteria in bile-salt-free (non-intestinal) environments is largely unknown. Here, we aimed to identify BSH from non-intestinal Enterococcus faecium and characterize its enzymatic function. We successfully isolated a plasmid-encoded bsh (efpBSH) from E. faecium, and the recombinant EfpBSH showed BSH activity that preferentially hydrolyzed taurine-conjugated bile salts, unlike the activity of known BSHs. EfpBSH functioned optimally at pH 4.0 and 50 °C. EfpBSH exhibited very low amino acid sequence similarity (48.46%) to EfBSH from E. faecalis T2 isolated from human urine, although 241 sequences with 100% identity to EfpBSH were found in both plasmids and chromosomes of E. faecium strains inhabiting intestinal and non-intestinal environments. Phylogenetically, EfpBSH was not affiliated with any known BSH phylogroup and was clearly distinguished from previously identified BSHs from intestinal lactic acid bacteria. Our genome database analysis demonstrated that horizontal gene transfer causes global efpBSH distribution among E. faecium strains in various environments (soil, water, and intestinal samples) and geographical regions (Asia, Africa, Europe, North America, South America, and Australia/Oceania). Overall, our findings are the first to indicate that BSH is not an intestine-specific enzyme and that hitherto-overlooked probiotic candidates with BSH activity can exist in diverse environments.
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Affiliation(s)
- Hiroyuki Kusada
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Ibaraki, Japan
| | - Hideyuki Tamaki
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Ibaraki, Japan
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2
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Karlov DS, Long SL, Zeng X, Xu F, Lal K, Cao L, Hayoun K, Lin J, Joyce SA, Tikhonova IG. Characterization of the mechanism of bile salt hydrolase substrate specificity by experimental and computational analyses. Structure 2023; 31:629-638.e5. [PMID: 36963397 DOI: 10.1016/j.str.2023.02.014] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2022] [Revised: 02/02/2023] [Accepted: 02/27/2023] [Indexed: 03/26/2023]
Abstract
Bile salt hydrolases (BSHs) are currently being investigated as target enzymes for metabolic regulators in humans and as growth promoters in farm animals. Understanding structural features underlying substrate specificity is necessary for inhibitor design. Here, we used a multidisciplinary workflow including mass spectrometry, mutagenesis, molecular dynamic simulations, machine learning, and crystallography to demonstrate substrate specificity in Lactobacillus salivarius BSH, the most abundant enzyme in human and farm animal intestines. We show the preference of substrates with a taurine head and a dehydroxylated sterol ring for hydrolysis. A regression model that correlates the relative rates of hydrolysis of various substrates in various enzyme mutants with the residue-substrate interaction energies guided the identification of structural determinants of substrate binding and specificity. In addition, we found T208 from another BSH protomer regulating the hydrolysis. The designed workflow can be used for fast and comprehensive characterization of enzymes with a broad range of substrates.
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Affiliation(s)
- Dmitry S Karlov
- School of Pharmacy, Medical Biology Centre, Queen's University Belfast, BT9 7BL Northern Ireland, UK
| | - Sarah L Long
- School of Biochemistry and Cell Biology, University College Cork, Cork T12 YT20, Ireland; APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland
| | - Ximin Zeng
- Department of Animal Science, The University of Tennessee, Knoxville, TN 37996, USA
| | - Fuzhou Xu
- Department of Animal Science, The University of Tennessee, Knoxville, TN 37996, USA; Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
| | - Kanhaya Lal
- School of Pharmacy, Medical Biology Centre, Queen's University Belfast, BT9 7BL Northern Ireland, UK
| | - Liu Cao
- Department of Animal Science, The University of Tennessee, Knoxville, TN 37996, USA
| | - Karim Hayoun
- School of Biochemistry and Cell Biology, University College Cork, Cork T12 YT20, Ireland; APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland
| | - Jun Lin
- Department of Animal Science, The University of Tennessee, Knoxville, TN 37996, USA.
| | - Susan A Joyce
- School of Biochemistry and Cell Biology, University College Cork, Cork T12 YT20, Ireland; APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland.
| | - Irina G Tikhonova
- School of Pharmacy, Medical Biology Centre, Queen's University Belfast, BT9 7BL Northern Ireland, UK.
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3
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Öztürk M, Kılıçsaymaz Z, Önal C. Site-Directed Mutagenesis of Bile Salt Hydrolase (BSH) from Lactobacillus plantarum B14 Confirms the Importance of the V58 and Y65 Amino Acids for Activity and Substrate Specificity. FOOD BIOTECHNOL 2023. [DOI: 10.1080/08905436.2022.2164299] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Affiliation(s)
- Mehmet Öztürk
- Department of Biology, Faculty of Arts and Science, Bolu Abant Izzet Baysal University, Bolu, Turkey
| | - Zekiye Kılıçsaymaz
- Department of Biology, Faculty of Arts and Science, Bolu Abant Izzet Baysal University, Bolu, Turkey
| | - Cansu Önal
- Department of Biology, Faculty of Arts and Science, Bolu Abant Izzet Baysal University, Bolu, Turkey
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Functional and Phylogenetic Diversity of BSH and PVA Enzymes. Microorganisms 2021; 9:microorganisms9040732. [PMID: 33807488 PMCID: PMC8066178 DOI: 10.3390/microorganisms9040732] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2021] [Revised: 03/24/2021] [Accepted: 03/29/2021] [Indexed: 12/12/2022] Open
Abstract
Bile salt hydrolase (BSH) and penicillin V acylase (PVA) are related enzymes that are classified as choloylglycine hydrolases (CGH). BSH enzymes have attracted significant interest for their ability to modulate the composition of the bile acid pool, alter bile acid signaling events mediated by the host bile acid receptors FXR and TGR5 and influence cholesterol homeostasis in the host, while PVA enzymes have been widely utilised in an industrial capacity in the production of semi-synthetic antibiotics. The similarities between BSH and PVA enzymes suggest common evolution of these enzymes and shared mechanisms for substrate binding and catalysis. Here, we compare BSH and PVA through analysis of the distribution, phylogeny and biochemistry of these microbial enzymes. The development of new annotation approaches based upon functional enzyme analyses and the potential implications of BSH enzymes for host health are discussed.
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Structural and enzymatic analysis of a dimeric cholylglycine hydrolase like acylase active on N-acyl homoserine lactones. Biochimie 2020; 177:108-116. [PMID: 32835734 DOI: 10.1016/j.biochi.2020.07.017] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2020] [Revised: 07/24/2020] [Accepted: 07/26/2020] [Indexed: 01/17/2023]
Abstract
The prevalence of substrate cross-reactivity between AHL acylases and β-lactam acylases provides a glimpse of probable links between quorum sensing and antibiotic resistance in bacteria. Both these enzyme classes belong to the N-terminal nucleophile (Ntn)-hydrolase superfamily. Penicillin V acylases alongside bile salt hydrolases constitute the cholylglycine hydrolase (CGH) group of the Ntn-hydrolase superfamily. Here we report the ability of two acylases, Slac1 and Slac2, from the marine bacterium Shewanella loihica-PV4 to hydrolyze AHLs. Three-dimensional structure of Slac1reveals the conservation of the Ntn hydrolase fold and CGH active site, making it a unique CGH exclusively active on AHLs. Slac1homologs phylogenetically cluster separate from reported CGHs and AHL acylases, thereby representing a functionally distinct sub-class of CGH that might have evolved as an adaptation to the marine environment. We hypothesize that Slac1 could provide the structural framework for understanding this subclass, and further our understanding of the evolutionary link between AHL acylases and β-lactam acylases.
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Das P, Marcišauskas S, Ji B, Nielsen J. Metagenomic analysis of bile salt biotransformation in the human gut microbiome. BMC Genomics 2019; 20:517. [PMID: 31234773 PMCID: PMC6591925 DOI: 10.1186/s12864-019-5899-3] [Citation(s) in RCA: 46] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2019] [Accepted: 06/12/2019] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND In the biochemical milieu of human colon, bile acids act as signaling mediators between the host and its gut microbiota. Biotransformation of primary to secondary bile acids have been known to be involved in the immune regulation of human physiology. Several 16S amplicon-based studies with inflammatory bowel disease (IBD) subjects were found to have an association with the level of fecal bile acids. However, a detailed investigation of all the bile salt biotransformation genes in the gut microbiome of healthy and IBD subjects has not been performed. RESULTS Here, we report a comprehensive analysis of the bile salt biotransformation genes and their distribution at the phyla level. Based on the analysis of shotgun metagenomes, we found that the IBD subjects harbored a significantly lower abundance of these genes compared to the healthy controls. Majority of these genes originated from Firmicutes in comparison to other phyla. From metabolomics data, we found that the IBD subjects were measured with a significantly low level of secondary bile acids and high levels of primary bile acids compared to that of the healthy controls. CONCLUSIONS Our bioinformatics-driven approach of identifying bile salt biotransformation genes predicts the bile salt biotransformation potential in the gut microbiota of IBD subjects. The functional level of dysbiosis likely contributes to the variation in the bile acid pool. This study sets the stage to envisage potential solutions to modulate the gut microbiome with the objective to restore the bile acid pool in the gut.
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Affiliation(s)
- Promi Das
- Department of Biology and Biological Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden
| | - Simonas Marcišauskas
- Department of Biology and Biological Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden
| | - Boyang Ji
- Department of Biology and Biological Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden
| | - Jens Nielsen
- Department of Biology and Biological Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden
- Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2800 Lyngby, Denmark
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Ru X, Zhang CC, Yuan YH, Yue TL, Guo CF. Bile salt hydrolase activity is present in nonintestinal lactic acid bacteria at an intermediate level. Appl Microbiol Biotechnol 2018; 103:893-902. [PMID: 30421106 DOI: 10.1007/s00253-018-9492-5] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2018] [Revised: 09/05/2018] [Accepted: 10/29/2018] [Indexed: 01/04/2023]
Abstract
It is generally considered that bile salt hydrolase (BSH) activity is hardly detected in nonintestinal lactic acid bacteria (LAB). The aim of this study was to investigate the distribution and intensity of BSH activity in LAB isolated from naturally fermented vegetables and milk. A total of 624 lactic acid bacterial strains classified into 6 genera and 50 species were isolated from 144 naturally fermented vegetable samples and 103 naturally fermented milk samples, and their BSH activity was screened by gas chromatography with electron capture detection. The BSH-positive strains were further analyzed quantitatively for their deconjugation ability against six human-conjugated bile salts by HPLC based on the disappearance of the conjugated bile salts from the reaction mixture. The results showed that 39% of the strains possessed BSH activity distributed in 24 lactic acid bacterial species. The strains of the fermented vegetable origin showed a 0.5-fold higher incidence of BSH-positive strains than those of the fermented milk origin, and the lactic acid bacilli exhibited 2.5-fold higher incidence of BSH-positive strains than the lactic acid cocci in general. The strains of the fermented vegetable origin generally had greater bile salt deconjugation ability than those of the fermented milk origin. More than 97% and 93% of the BSH-positive strains exhibited a greater substrate preference for glycoconjugated bile salts than tauroconjugated bile salts and for dihydroxy bile salts than trihydroxy bile salts, respectively. This study demonstrated that BSH activity was also present in nonintestinal LAB.
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Affiliation(s)
- Xiao Ru
- College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, China
| | - Chuang-Chuang Zhang
- College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, China
| | - Ya-Hong Yuan
- College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, China
| | - Tian-Li Yue
- College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, China
| | - Chun-Feng Guo
- College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, China.
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8
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Bustos AY, Font de Valdez G, Fadda S, Taranto MP. New insights into bacterial bile resistance mechanisms: the role of bile salt hydrolase and its impact on human health. Food Res Int 2018; 112:250-262. [DOI: 10.1016/j.foodres.2018.06.035] [Citation(s) in RCA: 60] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2018] [Revised: 05/14/2018] [Accepted: 06/18/2018] [Indexed: 01/18/2023]
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9
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Dong Z, Lee BH. Bile salt hydrolases: Structure and function, substrate preference, and inhibitor development. Protein Sci 2018; 27:1742-1754. [PMID: 30098054 DOI: 10.1002/pro.3484] [Citation(s) in RCA: 59] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2018] [Revised: 07/16/2018] [Accepted: 07/23/2018] [Indexed: 01/18/2023]
Abstract
The worldwide trend of limiting the use of antibiotic growth promoters (AGPs) in animal production creates challenges for the animal feed industry, thus necessitating the development of effective non-antibiotic alternatives to improve animal performance. Increasing evidence has shown that the growth-promoting effect of AGPs is highly correlated with the reduced activity of bile salt hydrolase (BSH, EC 3.5.1.24), an intestinal bacteria-producing enzyme that has a negative impact on host fat digestion and energy harvest. Therefore, BSH inhibitors may become novel, attractive alternatives to AGPs. Detailed knowledge of BSH substrate preferences and the wealth of structural data on BSHs provide a solid foundation for rationally tailored BSH inhibitor design. This review focuses on the relationship between structure and function of BSHs based on the crystal structure, kinetic data, molecular docking and comparative structural analyses. The molecular basis for BSH substrate recognition is also discussed. Finally, recent advances and future prospectives in the development of potent, safe, and cost-effective BSH inhibitors are described.
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Affiliation(s)
- Zixing Dong
- College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology, Tianjin, 300457, China
| | - Byong H Lee
- Department of Food Science and Biotechnology, Faculty of Agriculture and Life Sciences, Kangwon National University, Chuncheon, 200-701, South Korea.,Department of Microbiology/Immunology, McGill University, Montreal, Quebec, Canada, H3A 2B4
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10
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Yao L, Seaton SC, Ndousse-Fetter S, Adhikari AA, DiBenedetto N, Mina AI, Banks AS, Bry L, Devlin AS. A selective gut bacterial bile salt hydrolase alters host metabolism. eLife 2018; 7:e37182. [PMID: 30014852 PMCID: PMC6078496 DOI: 10.7554/elife.37182] [Citation(s) in RCA: 187] [Impact Index Per Article: 26.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2018] [Accepted: 07/06/2018] [Indexed: 02/07/2023] Open
Abstract
The human gut microbiota impacts host metabolism and has been implicated in the pathophysiology of obesity and metabolic syndromes. However, defining the roles of specific microbial activities and metabolites on host phenotypes has proven challenging due to the complexity of the microbiome-host ecosystem. Here, we identify strains from the abundant gut bacterial phylum Bacteroidetes that display selective bile salt hydrolase (BSH) activity. Using isogenic strains of wild-type and BSH-deleted Bacteroides thetaiotaomicron, we selectively modulated the levels of the bile acid tauro-β-muricholic acid in monocolonized gnotobiotic mice. B. thetaiotaomicron BSH mutant-colonized mice displayed altered metabolism, including reduced weight gain and respiratory exchange ratios, as well as transcriptional changes in metabolic, circadian rhythm, and immune pathways in the gut and liver. Our results demonstrate that metabolites generated by a single microbial gene and enzymatic activity can profoundly alter host metabolism and gene expression at local and organism-level scales.
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Affiliation(s)
- Lina Yao
- Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonUnited States
| | - Sarah Craven Seaton
- Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonUnited States
| | - Sula Ndousse-Fetter
- Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonUnited States
| | - Arijit A Adhikari
- Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonUnited States
| | - Nicholas DiBenedetto
- Department of Pathology, Massachusetts Host-Microbiome CenterBrigham and Women’s HospitalBostonUnited States
| | - Amir I Mina
- Division of Endocrinology, Diabetes and HypertensionBrigham and Women’s HospitalBostonUnited States
| | - Alexander S Banks
- Division of Endocrinology, Diabetes and HypertensionBrigham and Women’s HospitalBostonUnited States
| | - Lynn Bry
- Department of Pathology, Massachusetts Host-Microbiome CenterBrigham and Women’s HospitalBostonUnited States
| | - A Sloan Devlin
- Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonUnited States
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The Lactobacillus Bile Salt Hydrolase Repertoire Reveals Niche-Specific Adaptation. mSphere 2018; 3:3/3/e00140-18. [PMID: 29848760 PMCID: PMC5976879 DOI: 10.1128/msphere.00140-18] [Citation(s) in RCA: 79] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2018] [Accepted: 04/27/2018] [Indexed: 12/15/2022] Open
Abstract
Various Lactobacillus species have been reported to deconjugate bile acids in the gastrointestinal tract (GIT) through the action of bile salt hydrolase (BSH) proteins. This function contributes to altering the gut microbiota composition and bile metabolism and detoxification and to lowering cholesterol levels. Here, we investigated the Lactobacillus BSH repertoire across 170 sequenced species. We used hidden Markov models to distinguish between BSH and closely related penicillin-V acylase (PVA) proteins. Even though BSH and PVA proteins have very different target substrates, they share high sequence similarity and are often misannotated. We determined that 82/170 (48.24%) species encoded PVA proteins, 39/170 (22.94%) species encoded BSH proteins, and 8/170 (4.71%) species encoded both BSH and PVA proteins, while 57/170 (33.53%) species encoded neither. Mapping the occurrence of BSH-encoding species onto a phylogenetic tree revealed that BSH-encoding lactobacilli primarily adopt the vertebrate-adapted lifestyle but not the environmental or plant-associated subsets. Phylogenetic analysis of the BSH sequences revealed two distinct clades, several conserved motifs, and the presence of six previously reported active-site residues. These data will guide future mechanistic studies of BSH activity and contribute to the development and selection of BSH-encoding Lactobacillus strains with therapeutic potential.IMPORTANCE Bile acids play an integral role in shaping the gut microbiota and host physiology by regulating metabolic signaling, weight gain, and serum cholesterol and liver triglyceride levels. Given these important roles of bile acids, we investigated the presence of bile salt hydrolase (BSH) in Lactobacillus genomes representing 170 different species, determined strain- and species-specific patterns of occurrences, and expanded on the diversity of the BSH repertoire in this genus. While our data showed that 28% of Lactobacillus species encode BSH proteins, these species are associated mainly with vertebrate-adapted niches, demonstrating selective pressure on lactobacilli to evolve to adapt to specific environments. These new data will allow targeted selection of specific strains of lactobacilli and BSH proteins for future mechanistic studies to explore their therapeutic potential for treating metabolic disorders.
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12
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Structure and function of a highly active Bile Salt Hydrolase (BSH) from Enterococcus faecalis and post-translational processing of BSH enzymes. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2018; 1866:507-518. [PMID: 29325872 DOI: 10.1016/j.bbapap.2018.01.003] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/14/2017] [Revised: 11/02/2017] [Accepted: 01/04/2018] [Indexed: 12/14/2022]
Abstract
Bile Salt Hydrolase (BSH), a member of Cholylglycine hydrolase family, catalyzes the de-conjugation of bile acids and is evolutionarily related to penicillin V acylase (PVA) that hydrolyses a different substrate such as penicillin V. We report the three-dimensional structure of a BSH enzyme from the Gram-positive bacteria Enterococcus faecalis (EfBSH) which has manifold higher hydrolase activity compared to other known BSHs and displays unique allosteric catalytic property. The structural analysis revealed reduced secondary structure content compared to other known BSH structures, particularly devoid of an anti-parallel β-sheet in the assembly loop and part of a β-strand is converted to increase the length of a substrate binding loop 2. The analysis of the substrate binding pocket showed reduced volume owing to altered loop conformations and increased hydrophobicity contributed by a higher ratio of hydrophobic to hydrophilic groups present. The aromatic residues F18, Y20 and F65 participate in substrate binding. Thus, their mutation affects enzyme activity. Docking and Molecular Dynamics simulation studies showed effective polar complementarity present for the three hydroxyl (-OH) groups of GCA substrate in the binding site contributing to higher substrate specificity and efficient catalysis. These are unique features characteristics of this BSH enzyme and thought to contribute to its higher activity and specificity towards bile salts as well as allosteric effects. Further, mechanism of autocatalytic processing of Cholylglycine Hydrolases by the excision of an N-terminal Pre-peptide was examined by inserting different N-terminal pre-peptides in EfBSH sequence. The results suggest that two serine residues next to nucleophile cysteine are essential for autocalytic processing to remove precursor peptide. Since pre-peptide is absent in EfBSH the mutation of these serines is tolerated. This suggests that an evolution-mediated subordination of the pre-peptide excision site resulted in loss of pre-peptide in EfBSH and other related Cholylglycine hydrolases.
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13
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Long SL, Gahan CGM, Joyce SA. Interactions between gut bacteria and bile in health and disease. Mol Aspects Med 2017; 56:54-65. [PMID: 28602676 DOI: 10.1016/j.mam.2017.06.002] [Citation(s) in RCA: 334] [Impact Index Per Article: 41.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2016] [Revised: 05/26/2017] [Accepted: 06/07/2017] [Indexed: 01/18/2023]
Abstract
Bile acids are synthesized from cholesterol in the liver and released into the intestine to aid the digestion of dietary lipids. The host enzymes that contribute to bile acid synthesis in the liver and the regulatory pathways that influence the composition of the total bile acid pool in the host have been well established. In addition, the gut microbiota provides unique contributions to the diversity of bile acids in the bile acid pool. Gut microbial enzymes contribute significantly to bile acid metabolism through deconjugation and dehydroxylation reactions to generate unconjugated bile acids and secondary bile acids. These microbial enzymes (which include bile salt hydrolase (BSH) and bile acid-inducible (BAI) enzymes) are essential for bile acid homeostasis in the host and represent a vital contribution of the gut microbiome to host health. Perturbation of the gut microbiota in disease states may therefore significantly influence bile acid signatures in the host, especially in the context of gastrointestinal or systemic disease. Given that bile acids are ligands for host cell receptors (including the FXR, TGR5 and Vitamin D Receptor) alterations to microbial enzymes and associated changes to bile acid signatures have significant consequences for the host. In this review we examine the contribution of microbial enzymes to the process of bile acid metabolism in the host and discuss the implications for microbe-host signalling in the context of C. difficile infection, inflammatory bowel disease and other disease states.
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Affiliation(s)
- Sarah L Long
- APC Microbiome Institute, University College Cork, Cork, Ireland; School of Microbiology, University College Cork, Cork, Ireland
| | - Cormac G M Gahan
- APC Microbiome Institute, University College Cork, Cork, Ireland; School of Microbiology, University College Cork, Cork, Ireland; School of Pharmacy, University College Cork, Cork, Ireland.
| | - Susan A Joyce
- APC Microbiome Institute, University College Cork, Cork, Ireland; School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland
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14
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Chand D, Avinash VS, Yadav Y, Pundle AV, Suresh CG, Ramasamy S. Molecular features of bile salt hydrolases and relevance in human health. Biochim Biophys Acta Gen Subj 2017; 1861:2981-2991. [DOI: 10.1016/j.bbagen.2016.09.024] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Revised: 09/20/2016] [Accepted: 09/23/2016] [Indexed: 01/18/2023]
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15
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Sunder AV, Utari PD, Ramasamy S, van Merkerk R, Quax W, Pundle A. Penicillin V acylases from gram-negative bacteria degrade N-acylhomoserine lactones and attenuate virulence in Pseudomonas aeruginosa. Appl Microbiol Biotechnol 2016; 101:2383-2395. [DOI: 10.1007/s00253-016-8031-5] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2016] [Revised: 11/21/2016] [Accepted: 11/23/2016] [Indexed: 11/25/2022]
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16
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17
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Avinash VS, Panigrahi P, Chand D, Pundle A, Suresh CG, Ramasamy S. Structural analysis of a penicillin V acylase from Pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity. J Struct Biol 2015; 193:85-94. [PMID: 26707624 DOI: 10.1016/j.jsb.2015.12.008] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2015] [Revised: 12/16/2015] [Accepted: 12/16/2015] [Indexed: 10/22/2022]
Abstract
Penicillin V acylases (PVA) catalyze the deacylation of the beta-lactam antibiotic phenoxymethylpenicillin (Pen V). They are members of the Ntn hydrolase family and possess an N-terminal cysteine as the main catalytic nucleophile residue. They form the evolutionarily related cholylglycine hydrolase (CGH) group which includes bile salt hydrolases (BSH) responsible for bile deconjugation. Even though a few PVA and BSH structures have been reported, no structure of a functional PVA from Gram-negative bacteria is available. Here, we report the crystal structure of a highly active PVA from Gram-negative Pectobacterium atrosepticum (PaPVA) at 2.5Å resolution. Structural comparison with PVAs from Gram-positive bacteria revealed that PaPVA had a distinctive tetrameric structure and active site organization. In addition, mutagenesis of key active site residues and biochemical characterization of the resultant variants elucidated the role of these residues in substrate binding and catalysis. The importance of residue Trp23 and Trp87 side chains in binding and correct positioning of Pen V by PVAs was confirmed using mutagenesis and substrate docking with a 15ns molecular dynamics simulation. These results establish the unique nature of Gram-negative CGHs and necessitate further research about their substrate spectrum.
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Affiliation(s)
- Vellore Sunder Avinash
- Biochemical Sciences Division, National Chemical Laboratory (CSIR-NCL), Pune 411008, India
| | - Priyabrata Panigrahi
- Biochemical Sciences Division, National Chemical Laboratory (CSIR-NCL), Pune 411008, India
| | - Deepak Chand
- Biochemical Sciences Division, National Chemical Laboratory (CSIR-NCL), Pune 411008, India
| | - Archana Pundle
- Biochemical Sciences Division, National Chemical Laboratory (CSIR-NCL), Pune 411008, India
| | | | - Sureshkumar Ramasamy
- Biochemical Sciences Division, National Chemical Laboratory (CSIR-NCL), Pune 411008, India.
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Penicillin V acylase from Pectobacterium atrosepticum exhibits high specific activity and unique kinetics. Int J Biol Macromol 2015; 79:1-7. [PMID: 25931393 DOI: 10.1016/j.ijbiomac.2015.04.036] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2014] [Revised: 04/07/2015] [Accepted: 04/09/2015] [Indexed: 01/18/2023]
Abstract
Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria.
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Mukherji R, Prabhune A. Possible Correlation Between Bile Salt Hydrolysis and AHL Deamidation: Staphylococcus epidermidis RM1, a Potent Quorum Quencher and Bile Salt Hydrolase Producer. Appl Biochem Biotechnol 2015; 176:140-50. [DOI: 10.1007/s12010-015-1563-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2014] [Accepted: 03/12/2015] [Indexed: 11/29/2022]
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Gaci N, Borrel G, Tottey W, O’Toole PW, Brugère JF. Archaea and the human gut: New beginning of an old story. World J Gastroenterol 2014; 20:16062-16078. [PMID: 25473158 PMCID: PMC4239492 DOI: 10.3748/wjg.v20.i43.16062] [Citation(s) in RCA: 252] [Impact Index Per Article: 22.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/13/2014] [Revised: 05/14/2014] [Accepted: 07/22/2014] [Indexed: 02/06/2023] Open
Abstract
Methanogenic archaea are known as human gut inhabitants since more than 30 years ago through the detection of methane in the breath and isolation of two methanogenic species belonging to the order Methanobacteriales, Methanobrevibacter smithii and Methanosphaera stadtmanae. During the last decade, diversity of archaea encountered in the human gastrointestinal tract (GIT) has been extended by sequence identification and culturing of new strains. Here we provide an updated census of the archaeal diversity associated with the human GIT and their possible role in the gut physiology and health. We particularly focus on the still poorly characterized 7th order of methanogens, the Methanomassiliicoccales, associated to aged population. While also largely distributed in non-GIT environments, our actual knowledge on this novel order of methanogens has been mainly revealed through GIT inhabitants. They enlarge the number of final electron acceptors of the gut metabolites to mono- di- and trimethylamine. Trimethylamine is exclusively a microbiota-derived product of nutrients (lecithin, choline, TMAO, L-carnitine) from normal diet, from which seems originate two diseases, trimethylaminuria (or Fish-Odor Syndrome) and cardiovascular disease through the proatherogenic property of its oxidized liver-derived form. This therefore supports interest on these methanogenic species and its use as archaebiotics, a term coined from the notion of archaea-derived probiotics.
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