Characterization of a cold-adapted DNA photolyase from C. psychrerythraea 34H

Characterization of a cold-adapted DNA photolyase from C. psychrerythraea 34H The phrB gene encoding a putative cold-adapted DNA photolyase was cloned from the bacterial genomic DNA of Colwellia psychrerythraea 34H, a psychrophilic bacterium. Recombinant DNA photolyase, rCpPL, was overexpressed and purified from three different vectors. rCpPL binds its DNA substrate by flipping a cyclobutane pyrimidine dimer (CPD) into its active site and repairs CPD-containing DNA in vitro. rCpPL contains one catalytic flavin adenine dinucleotide (FAD) cofactor, but displays promiscuity in cofactor binding, in which either a flavin mononucleotide (FMN) or a methenyltetrahydrofolate (MTHF) molecule is bound as an antenna molecule and found in sub-stoichiometric amounts. The UV/Vis spectrum of oxidized rCpPL shows that the FADOX absorption maximum is the most red-shifted reported for a PL, suggesting a unique cavity electrostatic environment. Modest FAD vibronic structure suggests that the binding pocket is more flexible than warmer PLs, corroborating the hypothesis that psychrophilic proteins must be highly flexible to function at low temperatures. Fluorescence excitation data show that the freshly purified flavin cofactor is in its fully reduced state (FADH¯). A homology analysis of PL protein structures spanning 70 °C in growth temperature supports the data that the structure of CpPL is quite different from its warmer cousins. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Extremophiles Springer Journals

Characterization of a cold-adapted DNA photolyase from C. psychrerythraea 34H

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Publisher
Springer Japan
Copyright
Copyright © 2017 by Springer Japan KK
Subject
Life Sciences; Microbiology; Biotechnology; Biochemistry, general; Microbial Ecology
ISSN
1431-0651
eISSN
1433-4909
D.O.I.
10.1007/s00792-017-0953-z
Publisher site
See Article on Publisher Site

Abstract

The phrB gene encoding a putative cold-adapted DNA photolyase was cloned from the bacterial genomic DNA of Colwellia psychrerythraea 34H, a psychrophilic bacterium. Recombinant DNA photolyase, rCpPL, was overexpressed and purified from three different vectors. rCpPL binds its DNA substrate by flipping a cyclobutane pyrimidine dimer (CPD) into its active site and repairs CPD-containing DNA in vitro. rCpPL contains one catalytic flavin adenine dinucleotide (FAD) cofactor, but displays promiscuity in cofactor binding, in which either a flavin mononucleotide (FMN) or a methenyltetrahydrofolate (MTHF) molecule is bound as an antenna molecule and found in sub-stoichiometric amounts. The UV/Vis spectrum of oxidized rCpPL shows that the FADOX absorption maximum is the most red-shifted reported for a PL, suggesting a unique cavity electrostatic environment. Modest FAD vibronic structure suggests that the binding pocket is more flexible than warmer PLs, corroborating the hypothesis that psychrophilic proteins must be highly flexible to function at low temperatures. Fluorescence excitation data show that the freshly purified flavin cofactor is in its fully reduced state (FADH¯). A homology analysis of PL protein structures spanning 70 °C in growth temperature supports the data that the structure of CpPL is quite different from its warmer cousins.

Journal

ExtremophilesSpringer Journals

Published: Jul 19, 2017

References

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