Bacteroides thetaiotaomicron generates diverse α-mannosidase activities through subtle evolution of a distal substrate-binding motif

A dominant human gut microbe, the well studied symbiont Bacteroides thetaiotaomicron (Bt), is a glyco-specialist that harbors a large repertoire of genes devoted to carbohydrate processing. Despite strong similarities among them, many of the encoded enzymes have evolved distinct substrate specificities, and through the clustering of cognate genes within operons termed polysaccharide-utilization loci (PULs) enable the fulfilment of complex biological roles. Structural analyses of two glycoside hydrolase family 92 α-mannosidases, BT3130 and BT3965, together with mechanistically relevant complexes at 1.8–2.5 Å resolution reveal conservation of the global enzyme fold and core catalytic apparatus despite different linkage specificities. Structure comparison shows that Bt differentiates the activity of these enzymes through evolution of a highly variable substrate-binding region immediately adjacent to the active site. These observations unveil a genetic/biochemical mechanism through which polysaccharide-processing bacteria can evolve new and specific biochemical activities from otherwise highly similar gene products.

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Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

mBio ◽

10.1128/mbio.01120-13 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 31

Author(s):

Peter Mellroth ◽

Tatyana Sandalova ◽

Alexey Kikhney ◽

Francisco Vilaplana ◽

Dusan Hesek ◽

...

Keyword(s):

Cell Wall ◽

Streptococcus Pneumoniae ◽

Solution Structure ◽

Substrate Binding ◽

Substrate Recognition ◽

Lytic Activity ◽

Site Directed Mutagenesis ◽

Binding Motif ◽

Content Type ◽

Peptidoglycan Synthesis

ABSTRACT The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

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Structural Insights into 6-Hydroxypseudooxynicotine Amine Oxidase from Pseudomonas geniculata N1, the Key Enzyme Involved in Nicotine Degradation

Applied and Environmental Microbiology ◽

10.1128/aem.01559-20 ◽

2020 ◽

Vol 86 (19) ◽

Cited By ~ 1

Author(s):

Gongquan Liu ◽

Weiwei Wang ◽

Fangyuan He ◽

Peng Zhang ◽

Ping Xu ◽

...

Keyword(s):

Crystal Structure ◽

Structural Information ◽

Amine Oxidase ◽

Substrate Binding ◽

Docking Study ◽

Site Directed Mutagenesis ◽

Flavin Mononucleotide ◽

Structure Comparison ◽

Content Type ◽

Key Enzyme

ABSTRACT Bacteria degrade nicotine mainly using pyridine and pyrrolidine pathways. Previously, we discovered a hybrid of the pyridine and pyrrolidine pathways (the VPP pathway) in Pseudomonas geniculata N1 and characterized its key enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD). It catalyzes oxidative deamination of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde-pyridine, which is the crucial step connecting upstream and downstream portions of the VPP pathway. We determined the crystal structure of wild-type HisD to 2.6 Å. HisD is a monomer that contains a flavin mononucleotide, an iron-sulfur cluster, and ADP. On the basis of sequence alignment and structure comparison, a difference has been found among HisD, closely related trimethylamine dehydrogenase (TMADH), and histamine dehydrogenase (HADH). The flavin mononucleotide (FMN) cofactor is not covalently bound to any residue, and the FMN isoalloxazine ring is planar in HisD compared to TMADH or HADH, which forms a 6-S-cysteinyl flavin mononucleotide cofactor and has an FMN isoalloxazine ring in a “butterfly bend” conformation. Based on the structure, docking study, and site-directed mutagenesis, the residues Glu60, Tyr170, Asp262, and Trp263 may be involved in substrate binding. The expanded understanding of the substrate binding mode from this study may guide rational engineering of such enzymes for biodegradation of potential pollutants or for bioconversion to generate desired products. IMPORTANCE Nicotine is a major tobacco alkaloid in tobacco waste. Pyridine and pyrrolidine pathways are the two best-elucidated nicotine metabolic pathways; Pseudomonas geniculata N1 catabolizes nicotine via a hybrid between the pyridine and pyrrolidine pathways. The crucial enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD), links the upstream and downstream portions of the VPP pathway; however, there is little structural information about this important enzyme. In this study, we determined the crystal structure of HisD from Pseudomonas geniculata N1. Its basic insights about the structure may help us to guide the engineering of such enzymes for bioremediation and bioconversion applications.

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The human gut microbe Bacteroides thetaiotaomicron encodes the founding member of a novel glycosaminoglycan-degrading polysaccharide lyase family PL29

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004510 ◽

2018 ◽

Vol 293 (46) ◽

pp. 17906-17916 ◽

Cited By ~ 8

Author(s):

Didier Ndeh ◽

Jose Munoz Munoz ◽

Alan Cartmell ◽

David Bulmer ◽

Corinne Wills ◽

...

Keyword(s):

Dermatan Sulfate ◽

Microbial Interactions ◽

Bacteroides Thetaiotaomicron ◽

Human Gut ◽

Polysaccharide Lyase ◽

Founding Member ◽

Degrading Enzymes ◽

Biochemical Assays ◽

Gut Microbe ◽

Inducible Genes

Glycosaminoglycans (GAGs) and GAG-degrading enzymes have wide-ranging applications in the medical and biotechnological industries. The former are also an important nutrient source for select species of the human gut microbiota (HGM), a key player in host–microbial interactions. How GAGs are metabolized by the HGM is therefore of interest and has been extensively investigated in the model human gut microbe Bacteroides thetaiotaomicron. The presence of as-yet uncharacterized GAG-inducible genes in its genome and of related species, however, is testament to our incomplete understanding of this process. Nevertheless, it presents a potential opportunity for the discovery of additional GAG-degrading enzymes. Here, we investigated a gene of unknown function (BT_3328) from the chondroitin sulfate (CS) utilization locus of B. thetaiotaomicron. NMR and UV spectroscopic assays revealed that it encodes a novel polysaccharide lyase (PL), hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron (Bt)) and its ability to degrade the GAGs CS, dermatan sulfate (DS), and hyaluronic acid (HA). When incubated with HA, BtCDH generated a series of unsaturated HA sugars, including Δ4,5UA-GlcNAc, Δ4,5UA-GlcNAc-GlcA-GlcNac, Δ4,5UA-[GlcNAc-GlcA]2-GlcNac, and Δ4,5UA-[GlcNAc-GlcA]3-GlcNac, as end products and hence was classed as endo-acting. A combination of genetic and biochemical assays revealed that BtCDH localizes to the cell surface of B. thetaiotaomicron where it enables extracellular GAG degradation. BtCDH homologs were also detected in several other HGM species, and we therefore propose that it represents the founding member of a new polysaccharide lyase family (PL29). The current discovery also contributes new insights into CS metabolism by the HGM.

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Ascertaining the biochemical function of an essential pectin methylesterase in the gut microbe Bacteroides thetaiotaomicron

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014974 ◽

2020 ◽

Vol 295 (52) ◽

pp. 18625-18637

Author(s):

Cheng-Jie Duan ◽

Arnaud Baslé ◽

Marcelo Visona Liberato ◽

Joseph Gray ◽

Sergey A. Nepogodiev ◽

...

Keyword(s):

Gut Microbiota ◽

Critical Role ◽

Pectin Methylesterase ◽

Bacteroides Thetaiotaomicron ◽

Human Gut ◽

Human Gut Microbiota ◽

New Family ◽

Gut Microbe ◽

Dietary Nutrient ◽

Helical Proteins

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II–derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/β-hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed β-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella. Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.

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Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii

Scientific Reports ◽

10.1038/s41598-021-96659-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Cathleen Kmezik ◽

Daniel Krska ◽

Scott Mazurkewich ◽

Johan Larsbrink

Keyword(s):

Biochemical Characterization ◽

Glycoside Hydrolase Family ◽

Carbohydrate Active Enzymes ◽

Human Gut ◽

Glucuronoyl Esterase ◽

Cob Biomass ◽

Polysaccharide Utilization Loci ◽

Hydrolase Family ◽

Discrete Functions

AbstractBacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions—multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.

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Molecular dynamics insights into the structure, function, and substrate binding mechanism of mucin desulfating sulfatase of gut microbe Bacteroides fragilis

Journal of Cellular Biochemistry ◽

10.1002/jcb.26569 ◽

2018 ◽

Vol 119 (4) ◽

pp. 3618-3631 ◽

Cited By ~ 6

Author(s):

Ardhendu Bhusan Praharaj ◽

Budheswar Dehury ◽

Namita Mahapatra ◽

Shantanu Kumar Kar ◽

Santosh Kumar Behera

Keyword(s):

Molecular Dynamics ◽

Structure Function ◽

Substrate Binding ◽

Bacteroides Fragilis ◽

Binding Mechanism ◽

Gut Microbe

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The Common Partner of Several Methyltransferases TRMT112 Regulates the Expression of N6AMT1 Isoforms in Mammalian Cells

Biomolecules ◽

10.3390/biom9090422 ◽

2019 ◽

Vol 9 (9) ◽

pp. 422 ◽

Cited By ~ 1

Author(s):

Leetsi ◽

Õunap ◽

Abroi ◽

Kurg

Keyword(s):

Mammalian Cells ◽

Ribosome Biogenesis ◽

Substrate Binding ◽

Mrna Level ◽

Binding Motif ◽

Protein Levels ◽

Histone Lysine Methyltransferase ◽

Lysine Methyltransferase ◽

Alternatively Spliced ◽

The Common

Methylation is a widespread modification occurring in DNA, RNA and proteins. The N6AMT1 (HEMK2) protein has DNA N6-methyladenine as well as the protein glutamine and histone lysine methyltransferase activities. The human genome encodes two different isoforms of N6AMT1, the major isoform and the alternatively spliced isoform, where the substrate binding motif is missing. Several RNA methyltransferases involved in ribosome biogenesis, tRNA methylation and translation interact with the common partner, the TRMT112 protein. In this study, we show that TRMT112 regulates the expression of N6AMT1 isoforms in mammalian cells. Both isoforms are equally expressed on mRNA level, but only isoform 1 is detected on the protein level in human cells. We show that the alternatively spliced isoform is not able to interact with TRMT112 and when translated, is rapidly degraded from the cells. This suggests that TRMT112 is involved in cellular quality control ensuring that N6AMT1 isoform with missing substrate binding domain is eliminated from the cells. The down-regulation of TRMT112 does not affect the N6AMT1 protein levels in cells, suggesting that the two proteins of TRMT112 network, WBSCR22 and N6AMT1, are differently regulated by their common cofactor.

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Human resident gut microbe Bacteroides thetaiotaomicron regulates colonic neuronal innervation and neurogenic function

Gut Microbes ◽

10.1080/19490976.2020.1766936 ◽

2020 ◽

Vol 11 (6) ◽

pp. 1745-1757 ◽

Cited By ~ 3

Author(s):

Rubina Aktar ◽

Nabil Parkar ◽

Regis Stentz ◽

Lucas Baumard ◽

Aimee Parker ◽

...

Keyword(s):

Bacteroides Thetaiotaomicron ◽

Gut Microbe

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Structure and Characterization of Phosphoglucomutase 5 from Atlantic and Baltic Herring—An Inactive Enzyme with Intact Substrate Binding

Biomolecules ◽

10.3390/biom10121631 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1631

Author(s):

Robert Gustafsson ◽

Ulrich Eckhard ◽

Weihua Ye ◽

Erik D. Enbody ◽

Mats Pettersson ◽

...

Keyword(s):

Enzymatic Activity ◽

Active Site ◽

Muscle Protein ◽

Substrate Binding ◽

Clupea Harengus ◽

Metabolic Enzyme ◽

Structure Comparison ◽

Baltic Herring ◽

The Baltic

Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.

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