Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae

A gene encoding LgEstI was cloned from a bacterial fish pathogen, Lactococcus garvieae. Sequence and bioinformatic analysis revealed that LgEstI is close to the acetyl esterase family and had maximum similarity to a hydrolase (UniProt: Q5UQ83) from Acanthamoeba polyphaga mimivirus (APMV). Here, we present the results of LgEstI overexpression and purification, and its preliminary X-ray crystallographic analysis. The wild-type LgEstI protein was overexpressed in Escherichia coli, and its enzymatic activity was tested using p-nitrophenyl of varying lengths. LgEstI protein exhibited higher esterase activity toward p-nitrophenyl acetate. To better understand the mechanism underlying LgEstI activity and subject it to protein engineering, we determined the high-resolution crystal structure of LgEstI. First, the wild-type LgEstI protein was crystallized in 0.1 M Tris-HCl buffer (pH 7.1), 0.2 M calcium acetate hydrate, and 19% (w/v) PEG 3000, and the native X-ray diffraction dataset was collected up to 2.0 Å resolution. The crystal structure was successfully determined using a molecular replacement method, and structure refinement and model building are underway. The upcoming complete structural information of LgEstI may elucidate the substrate-binding mechanism and provide novel strategies for subjecting LgEstI to protein engineering.

Mechanism of the CBM35 domain in assisting catalysis by Ape1, a Campylobacter jejuni O-acetyl esterase

Peptidoglycan (PG) is O-acetylated by bacteria to resist killing by host lysozyme. During PG turnover, however, deacetylation is a prerequisite for glycan strand hydrolysis by lytic transglycosylases. Ape1, a de-O-acetylase from Campylobacter jejuni, is a bi-modular protein composed of an SGNH hydrolase domain and a CBM35 domain. The conserved Asp-His-Ser catalytic triad in the SGNH hydrolase domain confers enzymatic activity. The PG binding mode and function of the CBM35 domain in de-O-acetylation remained unclear. In this paper, we present a 1.8 Å resolution crystal structure of a complex between acetate and Ape1. An active site cleft is formed at the interface of the two domains and two large loops from the CBM35 domain form part of the active site. Site-directed mutagenesis of residues in these loops coupled with activity assays using p-nitrophenol acetate indicate the CBM35 loops are required for full catalytic efficiency. Molecular docking of a model O-acetylated hexasaccharide PG substrate to Ape1 using HADDOCK suggests the interaction is formed by the active cleft and the saccharide motif of PG. Together, we propose that the active cleft of Ape1 diverges from other SGNH hydrolase members by using the CBM35 loops to assist catalysis. The concave Ape1 active cleft may accommodate the long glycan strands for selecting PG substrates to regulate subsequent biological events.

Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans

Background Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized ‘treasure troves’ in the era of exponentially growing numbers of sequenced genomes. Results We determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/β-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs. Conclusions The bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.

Reducing enteric methane production from buffalo (Bubalus bubalis) by garlic oil supplementation in in vitro rumen fermentation system

Enteric methane production contributes significantly to the greenhouse gas emission globally. Although, buffaloes are integral part of livestock production in Asian countries, contributing milk, meat and draft power, the contribution of enteric methane to environmental pollution attracts attention. The present study investigated the efficacy of garlic (Allium sativum) oil in reducing enteric methane production from buffaloes (Bubalus bubalis) by in vitro rumen fermentation. Garlic oil (GOL) was tested at four concentrations [0 (Control), 33.33 µl (GOL-1), 83.33 µl (GOL-2) and 166.66 µl (GOL-3) per litre of buffered rumen fluid] in 100-ml graduated glass syringes and incubated at 39℃ for 24 h for in vitro rumen fermentation study. Supplementation of GOL-1 increased (p < 0.05) total gas production in comparison with GOL-3; however, it remained comparable (p > 0.05) with control and GOL-2. Graded doses of garlic oil inclusions reduced (p < 0.001) methane concentration (%) in total gas and total methane production (ml/g DM), irrespective of concentrations. The feed degradability, volatile fatty acids and microbial biomass production (MBP) were not affected (p > 0.05) by GOL-1, but these tended to decrease in GOL-2 with marked reduction (p < 0.01) in GOL-3. The decrease (p < 0.01) in NH3–N concentration in fermentation fluid in the presence of garlic oil, irrespective of concentration, suggests reduced deamination by inhibiting rumen proteolytic bacterial population. The activities of ruminal fibrolytic enzymes (CMCase, xylanase, β-glucosidase, acetyl esterase) were not affected by lower dose (GOL-1) of garlic oil; however, reduction (p < 0.05) of these enzymes activity in rumen liquor was evident at higher doses (GOL-2 and GOL-3) of supplementation. This study shows positive impact of garlic oil supplementation at low dose (33.33 µl/l of rumen fluid) in reducing enteric methane production, thereby, abatement of environmental pollution without affecting feed digestibility.

O-acetylated Gangliosides as Targets for Cancer Immunotherapy

O-acetylation of sialic acid residues is one of the main modifications of gangliosides, and modulates ganglioside functions. O-acetylation of gangliosides is dependent on sialyl-O-acetyltransferases and sialyl-O-acetyl-esterase activities. CAS1 Domain-Containing Protein 1 (CASD1) is the only human sialyl-O-acetyltransferases (SOAT) described until now. O-acetylated ganglioside species are mainly expressed during embryonic development and in the central nervous system in healthy adults, but are re-expressed during cancer development and are considered as markers of cancers of neuroectodermal origin. However, the specific biological roles of O-acetylated gangliosides in developing and malignant tissues have not been extensively studied, mostly because of the requirement of specific approaches and tools for sample preparation and analysis. In this review, we summarize our current knowledge of ganglioside biosynthesis and expression in normal and pathological conditions, of ganglioside O-acetylation analysis and expression in cancers, and of the possible use of O-acetylated gangliosides as targets for cancer immunotherapy.

ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of Enterococcus faecalis

Background ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis remains obscure. A clpP deletion mutant (△ clpP ) was constructed using the E. faecalis OG1RF strain to clarify the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with tandem mass tag labeling. Results The Δ clpP mutant strain showed impaired growth at 20°C or 45°C at 5% NaCl or 2 mM H 2 O 2 . The number of surviving Δ clpP mutants decreased after exposure to the high concentration (50× minimal inhibitory concentration) of linezolid or minocycline for 96 h. The Δ clpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 increased, 24 decreased) in the Δ clpP mutant strain. Among those, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx ( spxA ), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the Δ clpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein ( rplD ), ribosomal protein L7/L12 ( rplL2 ), 50S ribosomal protein L13 ( rplM ), L18 ( rplR ), L20 ( rplT ), 30S ribosomal protein S14 ( rpsN2 ) and S18 ( rpsR ) all decreased. The abundances of biofilm formation-related adapter protein MecA increased, while the abundances of dihydroorotase ( pyrC ), orotate phosphoribosyltransferase ( pyrE ), and orotidine-5'-phosphate decarboxylase ( pyrF ) all decreased in the Δ clpP mutant strain. Conclusion The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of E. faecalis.

ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of Enterococcus faecalis

Background ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis remains obscure. A clpP deletion mutant (△ clpP ) was constructed using the E. faecalis OG1RF strain to clarify the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with tandem mass tag labeling.Results The Δ clpP mutant strain showed impaired growth at 20°C or 45°C at 5% NaCl or 2 mM H 2 O 2 . The number of surviving Δ clpP mutants decreased after exposure to the high concentration (50× minimal inhibitory concentration) of linezolid or minocycline for 96 h. The Δ clpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 increased, 24 decreased) in the Δ clpP mutant strain. Among those, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx ( spxA ), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the Δ clpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein ( rplD ), ribosomal protein L7/L12 ( rplL2 ), 50S ribosomal protein L13 ( rplM ), L18 ( rplR ), L20 ( rplT ), 30S ribosomal protein S14 ( rpsN2 ) and S18 ( rpsR ) all decreased. The abundances of biofilm formation-related adapter protein MecA increased, while the abundances of dihydroorotase ( pyrC ), orotate phosphoribosyltransferase ( pyrE ), and orotidine-5'-phosphate decarboxylase ( pyrF ) all decreased in the Δ clpP mutant strain.Conclusion The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of E. faecalis.

Physiological significance of ClpP in Enterococcus faecalis and its regulating proteins were identified by Tandem Mass Tag Mass Spectrometry

Background ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis is still obscure. Thus a clpP deletion mutant (△clpP) was constructed in E. faecalis OG1RF strain to elucidate a more comprehensive picture of the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with Tandem Mass Tags labeling. Results The ΔclpP mutant strain showed impaired growth at 20°C or 45°C, at 5% NaCl or 2 mM H2O2. The surviving bacteria of the ΔclpP mutant strain reduced after exposure to the high concentration (50 x MIC) of linezolid or minocycline for 96 h. The ΔclpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 proteins increased, 24 proteins decreased) in the ΔclpP mutant strain. Among those differential abundance proteins, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx (spxA), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the ΔclpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein (rplD), ribosomal protein L7/L12 (rplL2), 50S ribosomal protein L13 (rplM), L18 (rplR), L20 (rplT), 30S ribosomal protein S14 (rpsN2) and S18 (rpsR) all reduced. The abundances of biofilm formation related adapter protein MecA increased, while the abundances of dihydroorotase (pyrC), orotate phosphoribosyltransferase (pyrE) and orotidine-5'-phosphate decarboxylase (pyrF) all decreased in the ΔclpP mutant strain. Conclusion The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobials tolerance, and virulence of E. faecalis.