Abstract
Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S](2+) cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S](+) state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5'-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5'-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.
Highlights
In bacteria and plants, activation of inorganic sulfur is required for de novo biosynthesis of cysteine
Under these conditions the yield of Mycobacterium tuberculosis adenosine 5-phosphosulfate reductase (MtAPR) was typically 7 mg/liter of culture, which represents an improvement over the ϳ1 mg/liter obtained when MtAPR is overexpressed in the absence of the isc proteins
The UV-visible absorbance spectrum of MtAPR showed a maximum in the visible range at 410 nm that is consistent with the presence of bound iron
Summary
APS and PAPS are reduced by enzymes in the reductive branch of the sulfate assimilation pathway, producing sulfite and AMP or adenosine 3Ј,5Ј-diphosphate (Scheme 1). These enzymes can be subdivided into two groups according to their substrate preference: the APS reductases (APR) and the PAPS reductases (PAPR) (EC 1.8.99.4). A key difference between the two enzymes is that APR contains two conserved cysteine motifs: CXXC and CC These four additional cysteine residues coordinate a [4Fe-4S] cluster, which is essential for catalytic activity [1, 5, 13].
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