Reversible resolution of flavin and pterin cofactors of His-tagged Escherichia coli DNA photolyase.
Escherichia coli photolyase catalyzes the repair of cyclobutane pyrimidine dimers (CPD) in DNA under near UV/blue-light irradiation. The enzyme contains flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF) as noncovalently bound light sensing cofactors. To study the apoprotein-chromophore interactions we developed a new procedure to prepare apo-photolyase. MTHF-free photolyase was obtained by binding the C-terminal His-tagged holoenzyme to a metal-affinity column at neutral pH and washing the column with deionized water. Under these conditions the flavin remains bound and the defolated enzyme can be released from the column with 0.5 M imidazole pH 7.2. The MTHF-free protein was still capable of DNA repair, showing 70% activity of native enzyme. Fluorescence polarization experiments confirmed that MTHF binding is weakened at low ionic strength. Apo-photolyase was obtained by treating the His-tagged holoenzyme with 0.5 M imidazole pH 10.0. The apo-photolyase thus obtained was highly reconstitutable and bound nearly stoichiometric amounts of FAD(ox). Photolyase reconstituted with FAD(ox) had about 34% activity of native enzyme, which increased to 83% when FAD(ox) was reduced to FADH(-). Reconstitution kinetics performed at 20 degrees C showed that apo-photolyase associates with FADH(-) much faster (k(obs) approximately 3,000 M(-1) s(-1)) than with FAD(ox) (k(obs)=16 [corrected] M(-1) s(-1)). The dissociation constant of the photolyase-FAD(ox) complex is about 2.3 microM and that of E-FADH(-) is not higher than 20 nM (pH 7.2).
Xu
Lei
L
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China. hsuley@ustc.edu
Zhang
Dongfang
D
Mu
Wanmeng
W
van Berkel
Willem J H
WJ
Luo
Zhaofeng
Z
D001051:Apoenzymes / Q000302:isolation & purification; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005454:Fluorescence Polarization; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D013053:Spectrophotometry; D013763:Tetrahydrofolates / Q000737:chemistry*
D001051:Apoenzymes; D013763:Tetrahydrofolates; D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; C018092:5,10-methenyltetrahydrofolate; D004255:Deoxyribodipyrimidine Photo-Lyase