Structural and biochemical studies of a moderately thermophilic exonuclease I from Methylocaldum szegediense

PLoS One. 2015 Feb 6;10(2):e0117470. doi: 10.1371/journal.pone.0117470. eCollection 2015.

Abstract

A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3' to 5' direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25-47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cloning, Molecular
  • Crystallography, X-Ray
  • DNA Repair / physiology
  • Exodeoxyribonucleases / chemistry*
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism
  • Hot Temperature
  • Methylococcaceae / enzymology*
  • Methylococcaceae / genetics

Substances

  • Bacterial Proteins
  • Exodeoxyribonucleases
  • exodeoxyribonuclease I

Grants and funding

The authors received no specific funding for this work. Li Fei, SiSi Tian, and Dapeng Sun are employees of New England Biolabs, Shanghai R&D Center. Tom Evans is an employee of New England Biolabs Inc. Ruth Moysey and Mihaela Misca are employees of Oxford Nanopore Technologies Ltd. John J. Barker, Myron A Smith, Paul A McEwan, Ewa S Pilka, and Lauren Crawley are employees of Evotec (UK) Ltd. New England Biolabs Shanghai R&D Center, Oxford Nanopore Technologies Ltd., Evotec (UK) Ltd and New England Biolabs provided support in the form of salaries, supplies, equipment, and infrastructure support for authors LF, ST, DS, TE,RM, MM, JJB, MAS, PAM, ESP and LC, provided editorial suggestions on content and data presentation for the manuscript and approved submission of the manuscript for publication, but did not have any additional role in control of the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.