TY - JOUR
T1 - Perchlorate reductase is distinguished by active site aromatic gate residues
AU - Youngblut, Matthew D.
AU - Tsai, Chi Lin
AU - Clark, Iain C.
AU - Carlson, Hans K.
AU - Maglaqui, Adrian P.
AU - Gau-Pan, Phonchien S.
AU - Redford, Steven A.
AU - Wong, Alan
AU - Tainer, John A.
AU - Coates, John D.
N1 - Funding Information:
We thank Dr. Joel Weiner's group from University of Alberta for generously providing the cytoplasm-facing nitrate reductase, NarGHI from E. coli (EccNarGHI). We thank SIBYLS beamline 12.3.1 staff scientist Scott Classen for assistance with crystallographic data collection. The crystallography experiments were conducted at the Advanced Light Source, a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies program supported by the Department of Energy Office of Biological and Environmental Research.
PY - 2016/4/22
Y1 - 2016/4/22
N2 - Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6/μM) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mM) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO32- bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.
AB - Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6/μM) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mM) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO32- bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.
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U2 - 10.1074/jbc.M116.714618
DO - 10.1074/jbc.M116.714618
M3 - Article
C2 - 26940877
AN - SCOPUS:84965047348
SN - 0021-9258
VL - 291
SP - 9190
EP - 9202
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 17
ER -