Alhosna Benjdia

A New Type of Bacterial Sulfatase Reveals a Novel Maturation Pathway in Prokaryotes

Sulfatases are a highly conserved family of enzymes found in all three domains of life. To be active, sulfatases undergo a unique post-translational modification leading to the conversion of either a critical cysteine (“Cys-type” sulfatases) or a serine (“Ser-type” sulfatases) into a Cα-formylglycine (FGly). This conversion depends on a strictly conserved sequence called “sulfatase signature” (C/S)XPXR. In a search for new enzymes from the human microbiota, we identified the first sulfatase from Firmicutes. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that this enzyme undergoes conversion of its critical cysteine residue into FGly, even though it has a modified (C/S)XAXR sulfatase signature. Examination of the bacterial and archaeal genomes sequenced to date has identified many genes bearing this new motif, suggesting that the definition of the sulfatase signature should be expanded. Furthermore, we have also identified a new Cys-type sulfatase-maturating enzyme that catalyzes the conversion of cysteine into FGly, in anaerobic conditions, whereas the only enzyme reported so far to be able to catalyze this reaction is oxygen-dependent. The new enzyme belongs to the radical S-adenosyl-l-methionine enzyme superfamily and is related to the Ser-type sulfatase-maturating enzymes. This finding leads to the definition of a new enzyme family of sulfatase-maturating enzymes that we have named anSME (anaerobic sulfatase-maturating enzyme). This family includes enzymes able to maturate Cys-type as well as Ser-type sulfatases in anaerobic conditions. In conclusion, our results lead to a new scheme for the biochemistry of sulfatases maturation and suggest that the number of genes and bacterial species encoding sulfatase enzymes is currently underestimated.

An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily

We report a catalytically promiscuous enzyme able to efficiently promote the hydrolysis of six different substrate classes. Originally assigned as a phosphonate monoester hydrolase (PMH) this enzyme exhibits substantial second-order rate accelerations ((kcat/KM)/kw), ranging from 107 to as high as 1019, for the hydrolyses of phosphate mono-, di-, and triesters, phosphonate monoesters, sulfate monoesters, and sulfonate monoesters. This substrate collection encompasses a range of substrate charges between 0 and -2, transition states of a different nature, and involves attack at two different reaction centers (P and S). Intrinsic reactivities (half-lives) range from 200 days to 105 years under near neutrality. The substantial rate accelerations for a set of relatively difficult reactions suggest that efficient catalysis is not necessarily limited to efficient stabilization of just one transition state. The crystal structure of PMH identifies it as a member of the alkaline phosphatase superfamily. PMH encompasses four of the native activities previously observed in this superfamily and extends its repertoire by two further activities, one of which, sulfonate monoesterase, has not been observed previously for a natural enzyme. PMH is thus one of the most promiscuous hydrolases described to date. The functional links between superfamily activities can be presumed to have played a role in functional evolution by gene duplication.

Thiostrepton tryptophan methyltransferase expands the chemistry of radical SAM enzymes

Methylation is among the most widespread chemical modifications encountered in biomolecules and has a pivotal role in many major biological processes. In the biosynthetic pathway of the antibiotic thiostrepton A, we identified what is to our knowledge the first tryptophan methyltransferase. We show that it uses unprecedented chemistry to methylate inactivated sp(2)-hybridized carbon atoms, despite being predicted to be a radical SAM enzyme.

Anaerobic Sulfatase-maturating Enzymes, First Dual Substrate Radical S-Adenosylmethionine Enzymes

Sulfatases are a major group of enzymes involved in many critical physiological processes as reflected by their broad distribution in all three domains of life. This class of hydrolases is unique in requiring an essential post-translational modification of a critical active-site cysteine or serine residue to Cα-formylglycine. This modification is catalyzed by at least three nonhomologous enzymatic systems in bacteria. Each enzymatic system is currently considered to be dedicated to the modification of either cysteine or serine residues encoded in the sulfatase-active site and has been accordingly categorized as Cys-type and Ser-type sulfatase-maturating enzymes. We report here the first detailed characterization of two bacterial anaerobic sulfatase-maturating enzymes (anSMEs) that are physiologically responsible for either Cys-type or Ser-type sulfatase maturation. The activity of both enzymes was investigated in vivo and in vitro using synthetic substrates and the successful purification of both enzymes facilitated the first biochemical and spectroscopic characterization of this class of enzyme. We demonstrate that reconstituted anSMEs are radical S-adenosyl-l-methionine enzymes containing a redox active [4Fe-4S]2+,+ cluster that initiates the radical reaction by binding and reductively cleaving S-adenosyl-l-methionine to yield 5 ′-deoxyadenosine and methionine. Surprisingly, our results show that anSMEs are dual substrate enzymes able to oxidize both cysteine and serine residues to Cα-formylglycine. Taken together, the results support a radical modification mechanism that is initiated by hydrogen abstraction from a serine or cysteine residue located in an appropriate target sequence.

Anaerobic sulfatase‐maturating enzyme – A mechanistic link with glycyl radical‐activating enzymes?

Sulfatases form a major group of enzymes present in prokaryotes and eukaryotes. This class of hydrolases is unique in requiring essential post‐translational modification of a critical active‐site cysteinyl or seryl residue to Cα‐formylglycine (FGly). Herein, we report mechanistic investigations of a unique class of radical‐S‐adenosyl‐L‐methionine (AdoMet) enzymes, namely anaerobic sulfatase‐maturating enzymes (anSMEs), which catalyze the oxidation of Cys‐type and Ser‐type sulfatases and possess three [4Fe‐4S]2+,+ clusters. We were able to develop a reliable quantitative enzymatic assay that allowed the direct measurement of FGly production and AdoMet cleavage. The results demonstrate stoichiometric coupling of AdoMet cleavage and FGly formation using peptide substrates with cysteinyl or seryl active‐site residues. Analytical and EPR studies of the reconstituted wild‐type enzyme and cysteinyl cluster mutants indicate the presence of three almost isopotential [4Fe‐4S]2+,+ clusters, each of which is required for the generation of FGly in vitro. More surprisingly, our data indicate that the two additional [4Fe‐4S]2+,+ clusters are required to obtain efficient reductive cleavage of AdoMet, suggesting their involvement in the reduction of the radical AdoMet [4Fe‐4S]2+,+ center. These results, in addition to the recent demonstration of direct abstraction by anSMEs of the Cβ H‐atom from the sulfatase active‐site cysteinyl or seryl residue using a 5′‐deoxyadenosyl radical, provide new insights into the mechanism of this new class of radical‐AdoMet enzymes.

Characterization of Glycosaminoglycan (GAG) Sulfatases from the Human Gut Symbiont Bacteroides thetaiotaomicron Reveals the First GAG-specific Bacterial Endosulfatase

Background: Sulfatases are emerging as key adaptive tools of commensal bacteria to their host. Results: The first bacterial endo-O-sulfatase and three exo-O-sulfatases from the human commensal Bacteroides thetaiotaomicron, specific for glycosaminoglycans, have been discovered and characterized. Conclusion: Commensal bacteria possess a unique array of highly specific sulfatases to metabolize host glycans. Significance: Bacterial sulfatases are much more diverse than anticipated. Despite the importance of the microbiota in human physiology, the molecular bases that govern the interactions between these commensal bacteria and their host remain poorly understood. We recently reported that sulfatases play a key role in the adaptation of a major human commensal bacterium, Bacteroides thetaiotaomicron, to its host (Benjdia, A., Martens, E. C., Gordon, J. I., and Berteau, O. (2011) J. Biol. Chem. 286, 25973–25982). We hypothesized that sulfatases are instrumental for this bacterium, and related Bacteroides species, to metabolize highly sulfated glycans (i.e. mucins and glycosaminoglycans (GAGs)) and to colonize the intestinal mucosal layer. Based on our previous study, we investigated 10 sulfatase genes induced in the presence of host glycans. Biochemical characterization of these potential sulfatases allowed the identification of GAG-specific sulfatases selective for the type of saccharide residue and the attachment position of the sulfate group. Although some GAG-specific bacterial sulfatase activities have been described in the literature, we report here for the first time the identity and the biochemical characterization of four GAG-specific sulfatases. Furthermore, contrary to the current paradigm, we discovered that B. thetaiotaomicron possesses an authentic GAG endosulfatase that is active at the polymer level. This type of sulfatase is the first one to be identified in a bacterium. Our study thus demonstrates that bacteria have evolved more sophisticated and diverse GAG sulfatases than anticipated and establishes how B. thetaiotaomicron, and other major human commensal bacteria, can metabolize and potentially tailor complex host glycans.

First evidences for a third sulfatase maturation system in prokaryotes from E. coli aslB and ydeM deletion mutants

To be active all known arylsulfatases undergo a unique post‐translational modification leading to the conversion of an active site residue (serine or cysteine) into a Cα‐formylglycine. Although deprived of sulfatase activity, Escherichia coli K12 can efficiently mature heterologous Cys‐type sulfatases. Three potential enzymes (AslB, YdeM and YidF) belonging to the anaerobic sulfatase maturating enzyme family (an SME) are present in its genome. Here we show that E. coli could mature Cys‐type sulfatases only in aerobic conditions and that knocking‐out of aslB, ydeM and yidF does not impair Cys‐type sulfatase maturation. These findings demonstrate that these putative anSME are not involved in Cys‐type sulfatase maturation and strongly support the existence of a second, oxygen‐dependent and Cys‐type specific sulfatase maturation system among prokaryotes.

The B12-Radical SAM Enzyme PoyC Catalyzes Valine Cβ-Methylation during Polytheonamide Biosynthesis

Genomic and metagenomic investigations have recently led to the delineation of a novel class of natural products called ribosomally synthesized and post-translationally modified peptides (RiPPs). RiPPs are ubiquitous among living organisms and include pharmaceutically relevant compounds such as antibiotics and toxins. A prominent example is polytheonamide A, which exhibits numerous post-translational modifications, some of which were unknown in ribosomal peptides until recently. Among these post-translational modifications, C-methylations have been proposed to be catalyzed by two putative radical S-adenosylmethionine (rSAM) enzymes, PoyB and PoyC. Here we report the in vitro activity of PoyC, the first B12-dependent rSAM enzyme catalyzing peptide Cβ-methylation. We show that PoyC catalyzes the formation of S-adenosylhomocysteine and 5′-deoxyadenosine and the transfer of a methyl group to l-valine residue. In addition, we demonstrate for the first time that B12-rSAM enzymes have a tightly bound MeCbl cofactor that during catalysis transfers a methyl group originating from S-adenosyl-l-methionine. Collectively, our results shed new light on polytheonamide biosynthesis and the large and emerging family of B12-rSAM enzymes.

Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis

Ribosomally synthesized peptides are built out of L-amino acids, whereas D-amino acids are generally the hallmark of non-ribosomal synthetic processes. Here we show that the model bacterium Bacillus subtilis is able to produce a novel type of ribosomally synthesized and post-translationally modified peptide that contains D-amino acids, and which we propose to call epipeptides. We demonstrate that a two [4Fe-4S]-cluster radical S-adenosyl-L-methionine (SAM) enzyme converts L-amino acids into their D-counterparts by catalysing Cα-hydrogen-atom abstraction and using a critical cysteine residue as the hydrogen-atom donor. Unexpectedly, these D-amino acid residues proved to be essential for the activity of a peptide that induces the expression of LiaRS, a major component of the bacterial cell envelope stress-response system. Present in B. subtilis and in several members of the human microbiome, these epipeptides and radical SAM epimerases broaden the landscape of peptidyl structures accessible to living organisms.

Chondroitin-4-O-sulfatase from Bacteroides thetaiotaomicron: exploration of the substrate specificity

Bacterial sulfatases can be good tools to increase the molecular diversity of glycosaminoglycan synthetic fragments. A chondroitin 4-O-sulfatase from the human commensal bacterium Bacteroides thetaiotaomicron has recently been identified and expressed. In order to use this enzyme for synthetic purposes, the minimal structure required for its activity has been determined. For that, four 4-O-sulfated monosaccharides and one 4-O-sulfated disaccharide have been synthesized and used as substrates with the sulfatase. The minimum structure was shown to be a disaccharide but in contrast to the natural substrate, which must have a 4,5-insaturation, the enzyme accepts as substrate, a disaccharide with a saturated glucuronic acid at the non-reducing end and even a glucopyranosyl moiety without the carboxylic acid functionality.