Function and Structure of a Prokaryotic Formylglycine-generating Enzyme

Brian L Carlson,Edward R Ballister,Emmanuel Skordalakes,David S King,Mark A Breidenbach,Sarah A Gilmore,James M Berger,Carolyn R Bertozzi

doi:10.1074/jbc.m800217200

Abstract

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 Cα-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 2.1Å 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.

Highlights

Motif [4] that defines this family of enzymes and is highly conserved throughout all domains of life (Fig. 1b)
The ions at m/z 1427 and 1445 are sodium adducts of the modified and unmodified peptide, respectively. b, upon treatment with biotin hydrazide, the formylglycyl residue (FGly)-containing peptide forms a hydrazone adduct with biotin, resulting in a mass shift of ϩ240 Da. c, lysates from wild-type (WT), ⌬fge, and complemented (⌬fge ϩ fge) strains of M. tuberculosis H37Rv were tested for sulfatase activity using the fluorogenic substrate 4-methylumbelliferyl sulfate (4MUS) with and without sulfatase/phosphatase inhibitors
Rv0712 was disrupted in M. tuberculosis H37Rv using homologous recombination and confirmed by Southern blot analysis. ⌬fge M. tuberculosis was viable and demonstrated no obvious growth defects in vitro

Summary

Introduction

Motif [4] that defines this family of enzymes and is highly conserved throughout all domains of life (Fig. 1b). B, upon treatment with biotin hydrazide, the FGly-containing peptide forms a hydrazone adduct with biotin, resulting in a mass shift of ϩ240 Da. c, lysates from wild-type (WT), ⌬fge, and complemented (⌬fge ϩ fge) strains of M. tuberculosis H37Rv were tested for sulfatase activity using the fluorogenic substrate 4-methylumbelliferyl sulfate (4MUS) with and without sulfatase/phosphatase inhibitors.

Results

Conclusion