PDBsum entry 2aik

Hydrolase activator,protein binding

PDB id: 2aik

Contents

Protein chain

274 a.a. *

Ligands

LEU-CYS-THR-PRO-SER-ARG-ALA

NAG-NAG

Metals

_CL

_CA ×2

Waters ×495

* Residue conservation analysis

PDB id:

2aik

Name:

Hydrolase activator,protein binding

Title:

Formylglycine generating enzyme c336s mutant covalently bound to substrate peptide lctpsra

Structure:

Sulfatase modifying factor 1. Chain: x. Fragment: residues 86-371. Synonym: c-alpha-formyglycine- generating enzyme 1. Engineered: yes. Mutation: yes. Lctpsra peptide from arylsulfatase a. Chain: p. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: homo sapiens. Expression_system_taxid: 9606. Expression_system_cell_line: ht1080. Expression_system_cell: fibrosarcoma cells. Synthetic: yes. Other_details: chemically synthesized

Biol. unit:

Dimer (from PQS)

Resolution:

1.73Å R-factor: 0.142 R-free: 0.174

Authors:

D.Roeser,M.G.Rudolph

Key ref:

D.Roeser et al. (2006). A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme. Proc Natl Acad Sci U S A, 103, 81-86. PubMed id: 16368756 DOI: 10.1073/pnas.0507592102

Date:

29-Jul-05 Release date: 13-Dec-05

Protein chain

Q8NBK3  (SUMF1_HUMAN) -  Formylglycine-generating enzyme from Homo sapiens

Seq: 374 a.a.

Struc: 274 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.8.3.7 - formylglycine-generating enzyme.
• Reaction: L-cysteinyl-[sulfatase] + 2 a thiol + O2 = an organic disulfide + 3-oxo- L-alanyl-[sulfatase] + hydrogen sulfide + H2O + H⁺
• Cofactor: Ca(2+); Cu cation

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Key reference

DOI no: 10.1073/pnas.0507592102

Proc Natl Acad Sci U S A 103:81-86 (2006)

PubMed id: 16368756

A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme.

D.Roeser, A.Preusser-Kunze, B.Schmidt, K.Gasow, J.G.Wittmann, T.Dierks, K.von Figura, M.G.Rudolph.

ABSTRACT

The formylglycine (FGly)-generating enzyme (FGE) uses molecular oxygen to oxidize a conserved cysteine residue in all eukaryotic sulfatases to the catalytically active FGly. Sulfatases degrade and remodel sulfate esters, and inactivity of FGE results in multiple sulfatase deficiency, a fatal disease. The previously determined FGE crystal structure revealed two crucial cysteine residues in the active site, one of which was thought to be implicated in substrate binding. The other cysteine residue partakes in a novel oxygenase mechanism that does not rely on any cofactors. Here, we present crystal structures of the individual FGE cysteine mutants and employ chemical probing of wild-type FGE, which defined the cysteines to differ strongly in their reactivity. This striking difference in reactivity is explained by the distinct roles of these cysteine residues in the catalytic mechanism. Hitherto, an enzyme-substrate complex as an essential cornerstone for the structural evaluation of the FGly formation mechanism has remained elusive. We also present two FGE-substrate complexes with pentamer and heptamer peptides that mimic sulfatases. The peptides isolate a small cavity that is a likely binding site for molecular oxygen and could host reactive oxygen intermediates during cysteine oxidation. Importantly, these FGE-peptide complexes directly unveil the molecular bases of FGE substrate binding and specificity. Because of the conserved nature of FGE sequences in other organisms, this binding mechanism is of general validity. Furthermore, several disease-causing mutations in both FGE and sulfatases are explained by this binding mechanism.

Selected figure(s)

Figure 2.
Substrate binding to FGE. (a) The surface representation of FGE shows a groove with the redox-active cysteine pair Cys-336/Cys-341 (red surface) at one end. Pro-182 (green surface) marks the site of a cross-link with a photoreactive substrate peptide (7) and hence is also close to the substrate binding site (8). (b) FGE–peptide complex. The peptide LCTPSRA binds to Cys-341 via an intermolecular disulfide bond. The FGE surface is colored according to electrostatic potential (±10 kT), showing a negative patch close to the C terminus of the peptide, which is neutralized by Arg-P73. (c) Close-up of b rotated 45° clockwise showing the exquisite surface complementarity of the peptide with FGE.

Figure 3.
Substrate binding and mechanistic details. (a) Hydrogen bonds are shown as dashed lines, and water molecules are drawn as red spheres. (b) General binding mechanism of FGE to all human sulfatases. The schematic drawing generalizes the binding of unfolded sulfatases to FGE as the first step in FGly formation. (c) Magnification of the region adjacent to the intermolecular disulfide bond. The orientation of the Tyr-340 side chain in the apo– and peptide–FGE structures differs by 6.4 Å (compare with Fig. 4). Only residues Cys-P69 and Thr-P70 of the peptide are drawn. The solvent-inaccessible volume between the disulfide bond and serine residues 333 and 336 (transparent gray sphere) is occupied by Cl^– (green) in the complex structure. (d) Possible mechanisms after the activation of molecular oxygen by FGE. Atoms from O[2] are indicated in red. A novel hydroperoxide intermediate is formulated from which two alternative avenues for FGly formation are conceivable. Currently, no distinction between these two pathways is possible.

Figures were selected by the author.