Sulfatases: Structure, Mechanism, Biological Activity, Inhibition, and Synthetic Utility

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134

The large-scale application of genomic and metagenomic sequencing technologies has yielded a number of insights about the metabolic potential of symbiotic human gut microbes. Nevertheless, the molecular basis of the interactions between commensal bacteria and their host remained to be investigated. Bacteria colonizing the mucosal layer that overlies the gut epithelium are exposed to highly sulfated glycans (i.e. mucin and glycosaminoglycans). These polymers can serve as potential nutrient sources, but their high sulfate content usually prevents their degradation. Commensal bacteria such as Bacteroides thetaiotaomicron possess more predicted sulfatase genes than in the human genome, the physiological functions of which are largely unknown. To be active, sulfatases must undergo a critical post-translational modification catalyzed in anaerobic bacteria by the radical AdoMet enzyme anaerobic sulfatase-maturating enzyme (anSME). In the present study, we have tested the role of this pathway in Bacteroides thetaiotaomicron which, in addition to 28 predicted sulfatases, possesses a single predicted anSME. In vitro studies revealed that deletion of the gene encoding its anSME (BT0238) results in loss of sulfatase activity and impaired ability to use sulfated polysaccharides as carbon sources. Co-colonization of formerly germ-free mice with both isogenic strains (i.e. wild-type or ⌬anSME), or invasion experiments involving introduction of one followed by the other strain established that anSME activity and the sulfatases activated via this pathway, are important fitness factors for B. thetaiotaomicron, especially when mice are fed a simple sugar diet that requires this saccharolytic bacterium to adaptively forage on host glycans as nutrients. Whole genome transcriptional profiling of wild-type and the anSME mutant in vivo revealed that loss of this enzyme alters expression of genes involved in mucin utilization and that this disrupted ability to access mucosal glycans likely underlies the observed pronounced colonization defect. Comparative genomic analysis reveals that 100% of 46 fully sequenced human gut Bacteroidetes contain homologs of BT0238 and genes encoding sulfatases, suggesting that this is an important and evolutionarily conserved feature for bacterial adaptation to life in this habitat.

Section: Identification Of Putative Sulfatases In Bacteroidesmentioning

confidence: 92%

Sulfatases and a Radical S-Adenosyl-l-methionine (AdoMet) Enzyme Are Key for Mucosal Foraging and Fitness of the Prominent Human Gut Symbiont, Bacteroides thetaiotaomicron

Benjdia

Martens

Gordon

et al. 2011

132

134

“…Type I sulfatases are members of an expanding family of enzymes that employ novel co-or post-translationally derived cofactors to facilitate catalysis (1,2). The formylglycine (Fgly) 4 residue positioned within the active site of type I sulfatases is thought to undergo hydration to a gem-diol, after which one of the hydroxyl groups acts as a catalytic nucleophile to initiate sulfate ester cleavage (Fig.…”

mentioning

confidence: 99%

“…In higher eukaryotes, sulfatases are involved in a variety of essential tasks, including extracellular matrix remodeling and steroid titer regulation (1). In humans, FGE deficiency abolishes the activity of all 17 human sulfatases, leading to the fatal disorder multiple sulfatase deficiency (6,15).…”

mentioning

confidence: 99%

“…Initial phases were determined by molecular replacement using the human FGE (Protein Data Bank entry 1Y1E) as a search model in PHASER (22). The asymmetric unit contained five S. coelicolor FGE monomers in space group P3 1 21. Initial stages of model refinement included cycles of simulated annealing with torsion angle dynamics and restrained B-factor refinement using CNS (23), followed by manual model rebuilding using O (24).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Function and Structure of a Prokaryotic Formylglycine-generating Enzyme

Carlson

Ballister

Skordalakes

et al. 2008

Type I sulfatases require an unusual co-or post-translational modification for their activity in hydrolyzing sulfate esters. In eukaryotic sulfatases, an active site cysteine residue is oxidized to the aldehyde-containing C ␣ -formylglycine residue by the formylglycine-generating enzyme (FGE). The machinery responsible for sulfatase activation is poorly understood in prokaryotes. Here we describe the identification of a prokaryotic FGE from Mycobacterium tuberculosis. In addition, we solved the crystal structure of the Streptomyces coelicolor FGE homolog to 2.1 Å resolution. The prokaryotic homolog exhibits remarkable structural similarity to human FGE, including the position of catalytic cysteine residues. Both biochemical and structural data indicate the presence of an oxidized cysteine modification in the active site that may be relevant to catalysis. In addition, we generated a mutant M. tuberculosis strain lacking FGE. Although global sulfatase activity was reduced in the mutant, a significant amount of residual sulfatase activity suggests the presence of FGE-independent sulfatases in this organism.Type I sulfatases are members of an expanding family of enzymes that employ novel co-or post-translationally derived cofactors to facilitate catalysis (1, 2). The formylglycine (Fgly) 4 residue positioned within the active site of type I sulfatases is thought to undergo hydration to a gem-diol, after which one of the hydroxyl groups acts as a catalytic nucleophile to initiate sulfate ester cleavage (Fig. 1a) (3). The FGly residue is located within a ϳ13-residue consensus sequence termed the sulfatase motif (4) that defines this family of enzymes and is highly conserved throughout all domains of life (Fig. 1b). Although FGly is formed from cysteine residues in eukaryotic sulfatases, either cysteine (within the core motif CX[P/A]XR) or serine (SXPXR) can be oxidized to FGly in prokaryotic type I sulfatases. The coor post-translational machineries necessary for these respective modifications appear to be different; FGE is able to activate CXPXR-type sulfatases (5-7) and anaerobic sulfatase-maturating enzyme is responsible for modifying SXPXR-type sulfatases and CXAXR-type sulfatases (8, 9). Some prokaryotes, such as Mycobacterium tuberculosis, have only CXPXR-type sulfatases (10), whereas other species have only SXPXR-type sulfatases or a combination of type I sulfatases (11). Some prokaryotes also contain FGly-independent sulfatases. These sulfatases function by distinct enzymatic mechanisms and are divided into two categories, Fe(II) ␣-ketoglutarate-dependent dioxygenase sulfatases (type II sulfatases) and metallo-␤-lactamase sulfatases (type III sulfatases) (12-14). Unlike type I sulfatases, which share a high degree of sequence similarity, type II and III sulfatases have highly divergent sequences, complicating the discovery of these proteins by genomic search algorithms.In higher eukaryotes, sulfatases are involved in a variety of essential tasks, including extracellular matrix remodeling and steroid titer regu...

“…The latter are found either at the cell surface (Sulf1, Sulf2), in the endoplasmic reticulum (arylsulfatases C, D, and F), or in the Golgi apparatus (arylsulfatase E) and act at neutral pH. In contrast, all lysosomal sulfatases (arylsulfatases A and B, iduronate-2-sulfatase, heparan-N-sulfatase, glucosamine-6-sulfatase, and galactosamine-6-sulfatase) share an acidic pH optimum (17). The genetic deficiency of each of these six lysosomal sulfatases causes specific and severe lysosomal storage disorders, namely metachromatic leukodystrophy and mucopolysaccharidoses type VI, II, IIIA, IIID, and IVA, respectively, which highlights the essential and non-redundant function of these enzymes (2).…”

mentioning

confidence: 99%

Arylsulfatase G, a Novel Lysosomal Sulfatase

Frese

Schulz²,

Dierks

2008

The sulfatases constitute a conserved family of enzymes that specifically hydrolyze sulfate esters in a wide variety of substrates such as glycosaminoglycans, steroid sulfates, or sulfolipids. By modifying the sulfation state of their substrates, sulfatases play a key role in the control of physiological processes, including cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked with various severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase G (ASG), was initially described as an enzyme lacking in vitro arylsulfatase activity and localizing to the endoplasmic reticulum. Contrary to these results, we demonstrate here that ASG does indeed have arylsulfatase activity toward different pseudosubstrates like p-nitrocatechol sulfate and 4-methylumbelliferyl sulfate. The activity of ASG depends on the Cys-84 residue that is predicted to be post-translationally converted to the critical active site C ␣ -formylglycine. Phosphate acts as a strong, competitive ASG inhibitor. ASG is active as an unprocessed 63-kDa monomer and shows an acidic pH optimum as typically seen for lysosomal sulfatases. In transfected cells, ASG accumulates within lysosomes as indicated by indirect immunofluorescence microscopy. Furthermore, ASG is a glycoprotein that binds specifically to mannose 6-phosphate receptors, corroborating its lysosomal localization. ARSG mRNA expression was found to be tissue-specific with highest expression in liver, kidney, and pancreas, suggesting a metabolic role of ASG that might be associated with a so far non-classified lysosomal storage disorder.