scholarly journals Molecular basis for catabolism of the abundant metabolite trans-4-hydroxy-L-proline by a microbial glycyl radical enzyme

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lindsey RF Backman ◽  
Yolanda Y Huang ◽  
Mary C Andorfer ◽  
Brian Gold ◽  
Ronald T Raines ◽  
...  
Keyword(s):  
Active Site ◽  
Metabolic Pathways ◽  
Molecular Basis ◽  
Radical Mechanism ◽  
Human Gut ◽  
Clostridioides Difficile ◽  
Glycyl Radical ◽  
Biochemical Studies ◽  
Glycyl Radical Enzyme ◽  
Insight Into

The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration.

scholarly journals Bioinformatic Characterization of Glycyl Radical Enzyme-Associated Bacterial Microcompartments

2015 ◽  
Vol 81 (24) ◽  
pp. 8315-8329 ◽  
Author(s):  
Jan Zarzycki ◽  
Onur Erbilgin ◽  
Cheryl A. Kerfeld
Keyword(s):  
Active Site ◽  
Metabolic Pathways ◽  
Experimental Investigations ◽  
Vitamin B ◽  
Bacterial Microcompartments ◽  
Glycyl Radical ◽  
Shell Composition ◽  
Glycyl Radical Enzyme ◽  
Assembly Pathways

ABSTRACTBacterial microcompartments (BMCs) are proteinaceous organelles encapsulating enzymes that catalyze sequential reactions of metabolic pathways. BMCs are phylogenetically widespread; however, only a few BMCs have been experimentally characterized. Among them are the carboxysomes and the propanediol- and ethanolamine-utilizing microcompartments, which play diverse metabolic and ecological roles. The substrate of a BMC is defined by its signature enzyme. In catabolic BMCs, this enzyme typically generates an aldehyde. Recently, it was shown that the most prevalent signature enzymes encoded by BMC loci are glycyl radical enzymes, yet little is known about the function of these BMCs. Here we characterize the glycyl radical enzyme-associated microcompartment (GRM) loci using a combination of bioinformatic analyses and active-site and structural modeling to show that the GRMs comprise five subtypes. We predict distinct functions for the GRMs, including the degradation of choline, propanediol, and fuculose phosphate. This is the first family of BMCs for which identification of the signature enzyme is insufficient for predicting function. The distinct GRM functions are also reflected in differences in shell composition and apparently different assembly pathways. The GRMs are the counterparts of the vitamin B12-dependent propanediol- and ethanolamine-utilizing BMCs, which are frequently associated with virulence. This study provides a comprehensive foundation for experimental investigations of the diverse roles of GRMs. Understanding this plasticity of function within a single BMC family, including characterization of differences in permeability and assembly, can inform approaches to BMC bioengineering and the design of therapeutics.

scholarly journals The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function

2019 ◽  
Vol 116 (41) ◽  
pp. 20404-20410 ◽  
Author(s):  
Amy J. Fernandez ◽  
Earnest James Paul Daniel ◽  
Sai Pooja Mahajan ◽  
Jeffrey J. Gray ◽  
Thomas A. Gerken ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Molecular Basis ◽  
Catalytic Domain ◽  
Binding Pocket ◽  
Substrate Selectivity ◽  
Peptide Substrate ◽  
X Ray ◽  
Biochemical Studies ◽  
Cancer Mutations ◽  
Insight Into

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.

scholarly journals Biochemical and structural characterization ofKlebsiella pneumoniaeoxamate amidohydrolase in the uric acid degradation pathway

2016 ◽  
Vol 72 (6) ◽  
pp. 808-816 ◽  
Author(s):  
Katherine A. Hicks ◽  
Steven E. Ealick
Keyword(s):  
Uric Acid ◽  
Active Site ◽  
Degradation Pathway ◽  
Α Subunit ◽  
Acid Degradation ◽  
Substrate Complex ◽  
Steady State Kinetics ◽  
Biochemical Studies ◽  
Kinetics Of ◽  
Insight Into

HpxW from the ubiquitous pathogenKlebsiella pneumoniaeis involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.

scholarly journals Pilot study: Trehalose-induced remodelling of the human microbiota affects Clostridioides difficile infection outcome in an in vitro colonic model

2021 ◽  
Author(s):  
Anthony M. Buckley ◽  
Ines B. Moura ◽  
Norie Arai ◽  
William Spittal ◽  
Emma Clark ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Intestinal Tract ◽  
Enhanced Recovery ◽  
Human Gut ◽  
Human Microbiota ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection ◽  
Saline Ingestion ◽  
Human Intestinal Tract

AbstractWithin the human intestinal tract, dietary, microbial- and host-derived compounds are used as signals by many pathogenic organisms, including Clostridioides difficile. Trehalose has been reported to enhance virulence of certain C. difficile ribotypes; however, such variants are widespread and not correlated with clinical outcomes for patients suffering from C. difficile infection (CDI). Here, we make preliminary observations to how to trehalose supplementation affects the microbiota in an in vitro model and show that trehalose can reduce the outgrowth of C. difficile, preventing simulated CDI. Three clinically reflective human gut models simulated the effects of sugar (trehalose or glucose) or saline ingestion on the microbiota. Models were instilled with sugar or saline and further exposed to C. difficile spores. The recovery of the microbiota following antibiotic treatment and CDI induction was monitored in each model. The human microbiota remodeled to utilise the bioavailable trehalose. Clindamycin induction caused simulated CDI in models supplemented with either glucose or saline; however, trehalose supplementation did not result in CDI, although limited spore germination did occur. The absence of CDI in trehalose model was associated with enhanced abundances of Finegoldia, Faecalibacterium and Oscillospira, and reduced abundances of Klebsiella and Clostridium spp., compared with the other models. Functional analysis of the microbiota in the trehalose model revealed differences in the metabolic pathways, such as amino acid metabolism, which could be attributed to prevention of CDI. Our data show that trehalose supplementation remodelled the microbiota, which prevented simulated CDI, potentially due to enhanced recovery of nutritionally competitive microbiota against C. difficile.

scholarly journals Molecular Basis of C–S Bond Cleavage in the Glycyl Radical Enzyme Isethionate Sulfite-Lyase

2020 ◽  
Author(s):  
Christopher D. Dawson ◽  
Stephania Irwin ◽  
Lindsey Backman ◽  
Catherine Drennan ◽  
Emily Balskus
Keyword(s):  
Molecular Basis ◽  
Bond Cleavage ◽  
Glycyl Radical ◽  
Glycyl Radical Enzyme

scholarly journals Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase

2016 ◽  
Vol 23 (10) ◽  
pp. 1206-1216 ◽  
Author(s):  
Smaranda Bodea ◽  
Michael A. Funk ◽  
Emily P. Balskus ◽  
Catherine L. Drennan
Keyword(s):  
Molecular Basis ◽  
Bond Cleavage ◽  
Glycyl Radical ◽  
Glycyl Radical Enzyme

scholarly journals A prominent glycyl radical enzyme in human gut microbiomes metabolizestrans-4-hydroxy-l-proline

Science ◽  
2017 ◽  
Vol 355 (6325) ◽  
pp. eaai8386 ◽  
Author(s):  
B. J. Levin ◽  
Y. Y. Huang ◽  
S. C. Peck ◽  
Y. Wei ◽  
A. Martínez-del Campo ◽  
...  
Keyword(s):  
Human Gut ◽  
Glycyl Radical ◽  
Glycyl Radical Enzyme

scholarly journals 3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Edward J Brignole ◽  
Kuang-Lei Tsai ◽  
Johnathan Chittuluru ◽  
Haoran Li ◽  
Yimon Aye ◽  
...  
Keyword(s):  
Active Site ◽  
Molecular Basis ◽  
Α Subunit ◽  
Cryo Electron Microscopy ◽  
Ribonucleotide Reductases ◽  
Dna Replication And Repair ◽  
Radical Transfer ◽  
Allosteric Regulators ◽  
Insight Into ◽  
Resolution Structure

Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the β subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α2 units arranged in an α6 ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α6 in the presence of both the anticancer drug clofarabine triphosphate and β2. Together, these structures support a model for RNR inhibition in which β2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.

Sign in / Sign up

Export Citation Format

Share Document