TY - JOUR
T1 - Escherichia coli Class Ib ribonucleotide reductase contains a dimanganese(III)-tyrosyl radical cofactor in vivo
AU - Cotruvo, Joseph A.
AU - Stubbe, Joanne
PY - 2011/3/15
Y1 - 2011/3/15
N2 - Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (Fe III2-Y•) and dimanganese(III)-Y • (MnIII2-Y•) cofactors in the β2 subunit, NrdF [Cotruvo, J. A., Jr., and Stubbe, J. (2010) Biochemistry 49, 1297-1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the MnIII2-Y• cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 μg of NrdF was isolated with ∼95% purity, with 0.2 Y •/β2, 0.9 Mn/β2, and a specific activity of 720 nmol min-1 mg-1. Under these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the MnIII 2-Y• cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo.
AB - Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (Fe III2-Y•) and dimanganese(III)-Y • (MnIII2-Y•) cofactors in the β2 subunit, NrdF [Cotruvo, J. A., Jr., and Stubbe, J. (2010) Biochemistry 49, 1297-1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the MnIII2-Y• cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 μg of NrdF was isolated with ∼95% purity, with 0.2 Y •/β2, 0.9 Mn/β2, and a specific activity of 720 nmol min-1 mg-1. Under these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the MnIII 2-Y• cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo.
UR - http://www.scopus.com/inward/record.url?scp=79952401377&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79952401377&partnerID=8YFLogxK
U2 - 10.1021/bi101881d
DO - 10.1021/bi101881d
M3 - Article
C2 - 21250660
AN - SCOPUS:79952401377
SN - 0006-2960
VL - 50
SP - 1672
EP - 1681
JO - Biochemistry
JF - Biochemistry
IS - 10
ER -