Mycobacterium tuberculosis Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging

Kimberly M Sogi,Mason J Appel,Carolyn R Bertozzi,Zev J Gartner,Michael W Schelle,Mark A Breidenbach

doi:10.1371/journal.pone.0065080

Abstract

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb’s single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase’s role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases.

Highlights

Sulfatases catalyze the cleavage of sulfate esters and are involved in diverse biological processes
We tested a panel of alkyl sulfate esters (Table 1) and found optimal activity on medium-chain substrates, 2-ethylhexyl sulfate (2-EHS) (Fig. 2A). n-Hexyl and n-heptyl sulfate showed moderate substrate activity, and n-pentyl sulfate was inefficiently desulfated (Fig. S1A)
Rv3406 was inactive on the aryl sulfate 4methylumbelliferyl sulfate (4-MUS) [21], a fluorogenic substrate often used in the study of type I sulfatases that lacks the requisite C-H bond that is cleaved in the type II sulfatase reaction

Summary

Introduction

Sulfatases catalyze the cleavage of sulfate esters and are involved in diverse biological processes. The roles of sulfatases in prokaryotes are less well defined, though most characterized bacterial genomes are predicted to encode at least one such enzyme. The human genome encodes only type I sulfatases, which cleave the RO–SO3– bond and consume one equivalent of water in the process. Two additional types of sulfatases have been identified in prokaryotic genomes. Type III sulfatases hydrolyze the same bond as do the type I enzymes, employing a Zn2+ cofactor to activate a nucleophilic water molecule using a mechanism related to that of some metalloproteases [10]. Type II sulfatases are nonheme iron-dependent dioxygenase that oxidize the C–H bond alpha to the sulfate ester using a-ketoglutarate (aKG) and oxygen as substrates (Fig. 1). The resulting hemiacetal sulfate ester collapses, liberating inorganic sulfate and an alkyl aldehyde, as well as water, carbon dioxide, and succinic acid byproducts [11]

Methods

Results

Conclusion