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 = 1/2 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1216-1226 |
| Number of pages | 11 |
| Journal | Journal of Biological Chemistry |
| Volume | 286 |
| Issue number | 2 |
| DOIs | |
| State | Published - Jan 14 2011 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
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Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5′-phosphosulfate reductase from Mycobacterium tuberculosis. / Bhave, Devayani P.; Hong, Jiyoung A.; Lee, Michael; Jiang, Wei; Krebs, Carsten; Carroll, Kate S.
In: Journal of Biological Chemistry, Vol. 286, No. 2, 14.01.2011, p. 1216-1226.Research output: Contribution to journal › Article
TY - JOUR
T1 - Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5′-phosphosulfate reductase from Mycobacterium tuberculosis
AU - Bhave, Devayani P.
AU - Hong, Jiyoung A.
AU - Lee, Michael
AU - Jiang, Wei
AU - Krebs, Carsten
AU - Carroll, Kate S.
PY - 2011/1/14
Y1 - 2011/1/14
N2 - 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 = 1/2 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.
AB - 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 = 1/2 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.
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U2 - 10.1074/jbc.M110.193722
DO - 10.1074/jbc.M110.193722
M3 - Article
C2 - 21075841
AN - SCOPUS:78651390685
VL - 286
SP - 1216
EP - 1226
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 2
ER -