Insights into the Glycyl Radical Enzyme Active Site of Benzylsuccinate Synthase: A Computational Study

2013 ◽  
Vol 135 (33) ◽  
pp. 12279-12288 ◽  
Author(s):  
Vivek S. Bharadwaj ◽  
Anthony M. Dean ◽  
C. Mark Maupin
Keyword(s):  
Active Site ◽  
Computational Study ◽  
Enzyme Active Site ◽  
Glycyl Radical ◽  
Benzylsuccinate Synthase ◽  
Glycyl Radical Enzyme

scholarly journals Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds

2004 ◽  
Vol 70 (5) ◽  
pp. 2935-2940 ◽  
Author(s):  
Barbara Morasch ◽  
Hans H. Richnow ◽  
Andrea Vieth ◽  
Bernhard Schink ◽  
Rainer U. Meckenstock
Keyword(s):  
Stable Isotope ◽  
Carbon Isotope ◽  
Isotope Fractionation ◽  
Toluene Degradation ◽  
Fractionation Factor ◽  
Glycyl Radical ◽  
Stable Isotope Fractionation ◽  
Benzylsuccinate Synthase ◽  
Glycyl Radical Enzyme

ABSTRACT Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors (ε) of −1.5 and −3.9, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic ε (εintrinsic) were calculated. A comparison of εintrinsic at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific ε elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of ε found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average ε if no fractionation factor is available for single compounds.

scholarly journals Structure and Function of Benzylsuccinate Synthase and Related Fumarate-Adding Glycyl Radical Enzymes

2016 ◽  
Vol 26 (1-3) ◽  
pp. 29-44 ◽  
Author(s):  
Johann Heider ◽  
Maciej Szaleniec ◽  
Berta M. Martins ◽  
Deniz Seyhan ◽  
Wolfgang Buckel ◽  
...  
Keyword(s):  
Anaerobic Metabolism ◽  
Biochemical Properties ◽  
Initial Structure ◽  
Toluene Degradation ◽  
Methyl Group ◽  
Glycyl Radical ◽  
Benzylsuccinate Synthase ◽  
And Function ◽  
Glycyl Radical Enzyme ◽  
Radical Type

The pathway of anaerobic toluene degradation is initiated by a remarkable radical-type enantiospecific addition of the chemically inert methyl group to the double bond of a fumarate cosubstrate to yield <i>(R)</i>-benzylsuccinate as the first intermediate, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. In recent years, it has become clear that benzylsuccinate synthase is the prototype enzyme of a much larger family of fumarate-adding enzymes, which play important roles in the anaerobic metabolism of further aromatic and even aliphatic hydrocarbons. We present an overview on the biochemical properties of benzylsuccinate synthase, as well as its recently solved structure, and present the results of an initial structure-based modeling study on the reaction mechanism. Moreover, we compare the structure of benzylsuccinate synthase with those predicted for different clades of fumarate-adding enzymes, in particular the paralogous enzymes converting <i>p</i>-cresol, 2-methylnaphthalene or <i>n</i>-alkanes.

scholarly journals Determinants for Substrate Recognition in the Glycyl Radical Enzyme Benzylsuccinate Synthase Revealed by Targeted Mutagenesis

ACS Catalysis ◽  
2021 ◽  
pp. 3361-3370
Author(s):  
Iryna Salii ◽  
Maciej Szaleniec ◽  
Ammar Alhaj Zein ◽  
Deniz Seyhan ◽  
Anna Sekuła ◽  
...  
Keyword(s):  
Substrate Recognition ◽  
Targeted Mutagenesis ◽  
Glycyl Radical ◽  
Benzylsuccinate Synthase ◽  
Glycyl Radical Enzyme

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 Benzylsuccinate Synthase is Post-Transcriptionally Regulated in the Toluene-Degrading Denitrifier Magnetospirillum sp. Strain 15-1

2020 ◽  
Vol 8 (5) ◽  
pp. 681 ◽  
Author(s):  
Ingrid Meyer-Cifuentes ◽  
Sylvie Gruhl ◽  
Sven-Bastiaan Haange ◽  
Vanessa Lünsmann ◽  
Nico Jehmlich ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Toluene Degradation ◽  
Regulation Of Expression ◽  
Glycyl Radical ◽  
Copy Numbers ◽  
Catalytically Active ◽  
Oxygen Sensitive ◽  
Benzylsuccinate Synthase ◽  
Glycyl Radical Enzyme ◽  
In Cells

The facultative denitrifying alphaproteobacterium Magnetospirillum sp. strain 15-1 had been isolated from the hypoxic rhizosphere of a constructed wetland model fed with toluene. This bacterium can catabolize toluene anaerobically but not aerobically. Here, we used strain 15-1 to investigate regulation of expression of the highly oxygen-sensitive glycyl radical enzyme benzylsuccinate synthase, which catalyzes the first step in anaerobic toluene degradation. In cells growing aerobically with benzoate, the addition of toluene resulted in a ~20-fold increased transcription of bssA, encoding for the catalytically active subunit of the enzyme. Under anoxic conditions, bssA mRNA copy numbers were up to 129-fold higher in cells growing with toluene as compared to cells growing with benzoate. Proteomics showed that abundance of benzylsuccinate synthase increased in cells growing anaerobically with toluene. In contrast, peptides of this enzyme were never detected in oxic conditions. These findings show that synthesis of benzylsuccinate synthase was under stringent post-transcriptional control in the presence of oxygen, which is a novel level of regulation for glycyl radical enzymes.

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.

Identification of FeS clusters in the glycyl-radical enzyme benzylsuccinate synthase via EPR and Mössbauer spectroscopy

2011 ◽  
Vol 17 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Markus Hilberg ◽  
Antonio J. Pierik ◽  
Eckhard Bill ◽  
Thorsten Friedrich ◽  
Marie-Luise Lippert ◽  
...  
Keyword(s):  
Mössbauer Spectroscopy ◽  
Mossbauer Spectroscopy ◽  
Mößbauer Spectroscopy ◽  
Glycyl Radical ◽  
Benzylsuccinate Synthase ◽  
Glycyl Radical Enzyme

scholarly journals Anaerobic Toluene Catabolism of Thauera aromatica: the bbs Operon Codes for Enzymes of β Oxidation of the Intermediate Benzylsuccinate

2000 ◽  
Vol 182 (2) ◽  
pp. 272-277 ◽  
Author(s):  
Birgitta Leuthner ◽  
Johann Heider
Keyword(s):  
Initial Reaction ◽  
Transcriptional Level ◽  
Beta Oxidation ◽  
Oxidation Pathway ◽  
Glycyl Radical ◽  
Thauera Aromatica ◽  
Coa Transferase ◽  
Benzylsuccinate Synthase ◽  
Glycyl Radical Enzyme ◽  
Induced Proteins

ABSTRACT The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent β-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in β oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting ofbbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the β-oxidation pathway of benzylsuccinate. Two of the genes, bbsE andbbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica. The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity. The further enzymes of the pathway are probably encoded bybbsH (enoyl-CoA hydratase), bbsCD(3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase). The operon contains two additional genes, bbsAand bbsI, for which no obvious function could be derived. The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the bbsA gene. This represents the first known promoter site in Thauera spp.

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