1.3 Å Structure of Arylsulfatase from Pseudomonas aeruginosa Establishes the Catalytic Mechanism of Sulfate Ester Cleavage in the Sulfatase Family

Boltes, Imke; Czapinska, H.; Kahnert, Antje; Bülow, Rixa von; Dierks, Thomas; Schmidt, Bernhard; Figura, Kurt Von; Kertesz, Michael A.; Usón, Isabel

doi:10.1016/s0969-2126(01)00609-8

Cited by 171 publications

(257 citation statements)

References 33 publications

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“…These structures all reveal a hydrolase fold similar to that of phosphonate monoester hydrolase (21) and arylsulfatase (22)(23)(24). A bound Zn 2+ ion is tetrahedrally coordinated by conserved residues (His453, Asp452, Glu240, and Thr280 in NmEptA).…”

Section: Significancementioning

confidence: 80%

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding

Anandan

Evans

Čondić‐Jurkić

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

128

231

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Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4′ headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different-sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.lipid modification | multidrug resistance | molecular dynamics | Neisseria | membrane protein structure

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Section: Significancementioning

confidence: 80%

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding

Anandan

Evans

Čondić‐Jurkić

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

128

231

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“…Aldehyde hydrates are generally unstable but nevertheless occur transiently in a number of reactions like that performed by alcohol dehydrogenase (63). They can even be stabilized like in arylsulfatases where a Ca 2ϩ -coordinated formyl hydrate originating from a conserved cystein or serine residue by posttranslational modification has been observed at the active site (64). The postulated formyl hydrate 6 could be stabilized by metal I in a similar way.…”

Section: Resultsmentioning

confidence: 99%

Structure of 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase from Methanococcus jannaschii in Complex with Divalent Metal Ions and the Substrate Ribulose 5-Phosphate

Steinbacher

Schiffmann

Richter

et al. 2003

Journal of Biological Chemistry

107

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Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-Å resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.Riboflavin (vitamin B 2 ) is biosynthesized in plants and numerous microorganisms but not in animals, which depend on nutritional sources. Its derivatives, flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD), are indispensable in all cells where they serve a variety of redox reactions (1). They have also been shown to serve a variety of other functions in cells such as DNA photorepair (2), light sensing (3, 4), and bioluminescence (5-8) and in reactions without net-redox change (9).Gram-negative bacteria are absolutely dependent on endogenous synthesis of riboflavin, because they are devoid of an uptake system for flavins or flavocoenzymes. Hence, riboflavindeficient mutants e.g. of Escherichia coli and Salmonella sp. require extremely high concentrations of exogenous riboflavin for growth. The same is true for yeasts such as Saccharomyces cerevisiae and Candida guilliermondii (for review see Refs. 1 and 10 -13). Thus, these organisms should be vulnerable to inhibitors of the riboflavin biosynthesis, which could therefore qualify as novel anti-infective agents. The absence of the riboflavin pathway in the human host appears advantageous in this context, because host/parasite selectivity of inhibitory agents would not constitute a problem. The mechanistic and structural analysis of the riboflavin pathway could serve as the basis for the rational design of riboflavin pathway inhibitors.3,4-Dihydroxy-2-butanone 4-phosphate synthase supplies the building blocks for the assembly of the xylene ring of the vitamin (14 -17). In fact, all eight carbon atoms of the xylene moiety are derived from the product of the enzyme. In the biosynthetic pathway, 3,4-dihydroxy-2-butanone 4-phosphate is condensed with 5-amino-6-ribitylamino-2,4(1H,3H)-...

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“…Pro-and eukaryotic sulfatases form a protein family whose members have similar structure and function (1)(2)(3)(4). Furthermore, they share a unique protein modification (5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

Characterization of Posttranslational Formylglycine Formation by Luminal Components of the Endoplasmic Reticulum

Fey¹,

Balleininger²,

Borissenko³

et al. 2001

Journal of Biological Chemistry

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C ␣ -formylglycine is the key catalytic residue in the active site of sulfatases. In eukaryotes formylglycine is generated during or immediately after sulfatase translocation into the endoplasmic reticulum by oxidation of a specific cysteine residue. We established an in vitro assay that allowed us to measure formylglycine modification independent of protein translocation. The modifying enzyme was recovered in a microsomal detergent extract. As a substrate we used ribosome-associated nascent chain complexes comprising in vitro synthesized sulfatase fragments that were released from the ribosomes by puromycin. Formylglycine modification was highly efficient and did not require a signal sequence in the substrate polypeptide. Ribosome association helped to maintain the modification competence of nascent chains but only after their release efficient modification occurred. The modifying machinery consists of soluble components of the endoplasmic reticulum lumen, as shown by differential extraction of microsomes. The in vitro assay can be performed under kinetically controlled conditions. The activation energy for formylglycine formation is 61 kJ/mol, and the pH optimum is Ϸ10. The activity is sensitive to the SH/SS equilibrium and is stimulated by Ca 2؉ . Formylglycine formation is efficiently inhibited by a synthetic sulfatase peptide representing the sequence directing formylglycine modification. The established assay system should make possible the biochemical identification of the modifying enzyme.

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1.3 Å Structure of Arylsulfatase from Pseudomonas aeruginosa Establishes the Catalytic Mechanism of Sulfate Ester Cleavage in the Sulfatase Family

Cited by 171 publications

References 33 publications

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding

Structure of 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase from Methanococcus jannaschii in Complex with Divalent Metal Ions and the Substrate Ribulose 5-Phosphate

Characterization of Posttranslational Formylglycine Formation by Luminal Components of the Endoplasmic Reticulum

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