4-hydroxyphenylacetate Decarboxylase Research Articles

Culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR.

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions employing antibiotics. This suggests a significant bias among analyzed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA-operon and thereby identifies low abundances of C. difficile in environmental samples. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para-cresol. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessment of C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research.IMPORTANCEClostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies, however, require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoidance of antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile-specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using next-generation sequencing.

Lateral gene transfer of p-cresol- and indole-producing enzymes from environmental bacteria to Mastigamoeba balamuthi.

p-Cresol and indole are volatile biologically active products of the bacterial degradation of tyrosine and tryptophan respectively. They are typically produced by bacteria in animal intestines, soil and various sediments. Here, we demonstrate that the free-living eukaryote Mastigamoeba balamuthi and its pathogenic relative Entamoeba histolytica produce significant amounts of indole via tryptophanase activity. Unexpectedly, M. balamuthi also produces p-cresol in concentrations that are bacteriostatic to non-p-cresol-producing bacteria. The ability of M.balamuthi to produce p-cresol, which has not previously been observed in any eukaryotic microbe, was gained due to the lateral acquisition of a bacterial gene for 4-hydroxyphenylacetate decarboxylase (HPAD). In bacteria, the genes for HPAD and the S-adenosylmethionine-dependent activating enzyme (AE) are present in a common operon. In M. balamuthi, HPAD displays a unique fusion with the AE that suggests the operon-mediated transfer of genes from a bacterial donor. We also clarified that the tyrosine-to-4-hydroxyphenylacetate conversion proceeds via the Ehrlich pathway. The acquisition of the bacterial HPAD gene may provide M. balamuthi a competitive advantage over other microflora in its native habitat.

A mechanistic study of the non-oxidative decarboxylation catalyzed by the radical S-adenosyl-l-methionine enzyme BlsE involved in blasticidin S biosynthesis

Decarboxylation is a fundamentally important reaction in biology and involves highly diverse mechanisms. Here we report a mechanistic study of the non-oxidative decarboxylation catalyzed by BlsE, a radical S-adenosyl-l-methionine (SAM) enzyme involved in blasticidin S biosynthesis. Through a series of biochemical analysis with isotopically labeled reagents, we show that the BlsE-catalyzed reaction is initiated by the 5'-deoxyadenosyl (dAdo) radical-mediated hydrogen abstraction from a sugar carbon of the substrate cytosylglucuronic acid (CGA), and does not involve a carboxyl radical as has been proposed for 4-hydroxyphenylacetate decarboxylase (HPAD). Our study reveals that BlsE represents a mechanistically new type of radical-based decarboxylase.

PyCPR - a python-based implementation of the Conjugate Peak Refinement (CPR) algorithm for finding transition state structures.

Conjugate peak refinement (CPR) is a powerful and robust method to search transition states on a molecular potential energy surface. Nevertheless, the method was to the best of our knowledge so far only implemented in CHARMM. In this paper, we present PyCPR, a new Python-based implementation of the CPR algorithm within the pDynamo framework. We provide a detailed description of the theory underlying our implementation and discuss the different parts of the implementation. The method is applied to two different problems. First, we illustrate the method by analyzing the gauche to anti-periplanar transition of butane using a semiempirical QM method. Second, we reanalyze the mechanism of a glycyl-radical enzyme, namely of 4-hydroxyphenylacetate decarboxylase (HPD) using QM/MM calculations. In the end, we suggest a strategy how to use our implementation of the CPR algorithm. The integration of PyCPR into the framework pDynamo allows the combination of CPR with the large variety of methods implemented in pDynamo. PyCPR can be used in combination with quantum mechanical and molecular mechanical methods (and hybrid methods) implemented directly in pDynamo, but also in combination with external programs such as ORCA using pDynamo as interface. PyCPR is distributed as free, open source software and can be downloaded from http://www.bisb.uni-bayreuth.de/index.php?page=downloads . Graphical Abstract PyCPR is a search tool for finding saddle points on the potential energy landscape of a molecular system.

The ferredoxin-like domain of the activating enzyme is required for generating a lasting glycyl radical in 4-hydroxyphenylacetate decarboxylase.

4-Hydroxyphenylacetate decarboxylase-activating enzyme (4Hpad-AE) uses S-adenosylmethionine (SAM or AdoMet) and a [4Fe-4S] ²⁺/⁺cluster (RS cluster) to generate a stable glycyl radical on the decarboxylase. 4Hpad-AE might bind up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert C-terminal to the RS cluster-binding motif. Except for the AEs of pyruvate formate-lyase and anaerobic ribonucleotide reductase, all glycyl radical-activating enzymes possess a similar ferredoxin-like domain, whose functional role is still poorly understood. To assess the role of the putative ferredoxin clusters from 4Hpad-AE, we combined biochemical and spectroscopic methods to characterize a truncated version of the protein (Δ66-AE) devoid of the ferredoxin-like domain. We found that Δ66-AE is stable, harbors a fully active RS cluster and can activate the decarboxylase. From the similar cleavage rates for S-adenosylmethionine of Δ66-AE and wild-type AE, we infer the reactivity of the RS cluster is unperturbed by the absence of the ferredoxin-like domain. Thus, the auxiliary clusters are not required as electron conduit to the RS cluster for effective reductive cleavage of SAM. The activation of the decarboxylase by Δ66-AE is almost as fast as with wild-type AE, but the generated glycyl radical is short living. We postulate that the ferredoxin-like domain is not required for SAM-dependent glycyl radical generation in the decarboxylase, but is necessary for producing a lasting glycyl radical.

Catalytic Mechanism of the Glycyl Radical Enzyme 4-Hydroxyphenylacetate Decarboxylase from Continuum Electrostatic and QC/MM Calculations

Using continuum electrostatics and QC/MM calculations, we investigate the catalytic cycle of the glycyl radical enzyme 4-hydroxyphenylacetate decarboxylase, an enzyme involved in the fermentative production of p-cresol from tyrosine in clostridia. On the basis of our calculations, we propose a five-step mechanism for the reaction. In the first step, the substrate 4-hydroxyphenylacetate is activated by an unusual concerted abstraction of an electron and a proton. Namely, Cys503 radical abstracts an electron from the substrate and Glu637 abstracts a proton. Thus in total, a hydrogen atom is abstracted from the substrate. In the second step, the carboxylic group readily splits off from the phenoxy-acetate radical anion to give carbon dioxide. This decarboxylation step is coupled to a proton transfer from Glu637 back to the phenolic hydroxyl group which leads to a p-hydroxybenzyl radical. The remaining steps of the reaction involve a rotation of the Cys503 side chain followed by a proton transfer from Glu505 to Cys503 and a hydrogen atom transfer from Cys503 to the p-hydroxybenzyl radical to form p-cresol. The calculated mechanism agrees with experimental data suggesting that both Cys503 and Glu637 are essential for the catalytic function of 4-hydroxyphenylacetate decarboxylase and that the substrate requires a hydroxyl group in para-position to the acetate moiety.

4-Hydroxyphenylacetate decarboxylase activating enzyme catalyses a classical S-adenosylmethionine reductive cleavage reaction

4-Hydroxyphenylacetate decarboxylase (4Hpad) is an Fe/S cluster containing glycyl radical enzyme (GRE), which catalyses the last step of tyrosine fermentation in clostridia, generating the bacteriostatic p-cresol. The respective activating enzyme (4Hpad-AE) displays two cysteine-rich motifs in addition to the classical S-adenosylmethionine (SAM) binding cluster (RS cluster) motif. These additional motifs are also present in other glycyl radical activating enzymes (GR-AE) and it has been postulated that these orthologues may use an alternative SAM homolytic cleavage mechanism, generating a putative 3-amino-3-carboxypropyl radical and 5'-deoxy-5'-(methylthio)adenosine but not a 5'-deoxyadenosyl radical and methionine. 4Hpad-AE produced from a codon-optimized synthetic gene binds a maximum of two [4Fe-4S](2+/+) clusters as revealed by EPR and Mössbauer spectroscopy. The enzyme only catalyses the turnover of SAM under reducing conditions, and the reaction products were identified as 5'-deoxyadenosine (quenched form of 5'-deoxyadenosyl radical) and methionine. We demonstrate that the 5'-deoxyadenosyl radical is the activating agent for 4Hpad through p-cresol formation and correlation between the production of 5'-deoxyadenosine and the generation of glycyl radical in 4Hpad. Therefore, we conclude that 4Hpad-AE catalyses a classical SAM-dependent glycyl radical formation as reported for GR-AE without auxiliary clusters. Our observation casts doubt on the suggestion that GR-AE containing auxiliary clusters catalyse the alternative cleavage reaction detected for glycerol dehydratase activating enzyme.

Structural Basis for a Kolbe-Type Decarboxylation Catalyzed by a Glycyl Radical Enzyme

4-Hydroxyphenylacetate decarboxylase is a [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in clostridia. The decarboxylation product p-cresol (4-methylphenol) is a virulence factor of the human pathogen Clostridium difficile . Here we describe the crystal structures at 1.75 and 1.81 Å resolution of substrate-free and substrate-bound 4-hydroxyphenylacetate decarboxylase from the related Clostridium scatologenes . The structures show a (βγ)(4) tetramer of heterodimers composed of a catalytic β-subunit harboring the putative glycyl/thiyl dyad and a distinct small γ-subunit with two [4Fe-4S] clusters at 40 Å distance from the active site. The γ-subunit comprises two domains displaying pseudo-2-fold symmetry that are structurally related to the [4Fe-4S] cluster-binding scaffold of high-potential iron-sulfur proteins. The N-terminal domain coordinates one cluster with one histidine and three cysteines, and the C-terminal domain coordinates the second cluster with four cysteines. Whereas the C-terminal cluster is buried in the βγ heterodimer interface, the N-terminal cluster is not part of the interface. The previously postulated decarboxylation mechanism required the substrate's hydroxyl group in the vicinity of the active cysteine residue. In contrast to expectation, the substrate-bound state shows a direct interaction between the substrate's carboxyl group and the active site Cys503, while His536 and Glu637 at the opposite side of the active site pocket anchor the hydroxyl group. This state captures a possible catalytically competent complex and suggests a Kolbe-type decarboxylation for p-cresol formation.

New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria

During the last decade, an increasing number of new enzymes containing glycyl radicals in their active sites have been identified and biochemically characterised. These include benzylsuccinate synthase (Bss), 4-hydroxyphenylacetate decarboxylase (Hpd) and the coenzyme B12-independent glycerol dehydratase (Gdh). These are involved in metabolic pathways as different as anaerobic toluene metabolism, fermentative production of p-cresol and glycerol fermentation. Some features of these newly discovered enzymes are described and compared with those of the previously known glycyl radical enzymes pyruvate formate-lyase (Pfl) and anaerobic ribonucleotide reductase (Nrd). Among the new enzymes, Bss and Hpd share the presence of small subunits, the function of which in the catalytic mechanisms is still enigmatic, and both enzymes contain metal centres in addition to the glycyl radical prosthetic group. The activating enzymes of the novel systems also deviate from the standard type, containing at least one additional Fe-S cluster. Finally, the available whole-genome sequences of an increasing number of strictly or facultative anaerobic bacteria revealed the presence of many more hitherto unknown glycyl radical enzyme (GRE) systems. Recent studies suggest that the particular types of these enzymes represent the ends of different evolutionary lines, which emerged early in evolution and diversified to yield remarkably versatile biocatalysts for chemical reactions that are otherwise difficult to perform in anoxic environments.