An unusual light-sensing function for coenzyme B12 in bacterial transcription regulator CarH

Harshwardhan Poddar, Derren J. Heyes, Shaowei Zhang, Samantha J. Hardman, Michiyo Sakuma, Nigel S. Scrutton

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

Coenzyme B12 is one of the most complex cofactors found in nature and synthesized de novo by certain groups of bacteria. Although its use in various enzymatic reactions is well characterized, only recently an unusual light-sensing function has been ascribed to coenzyme B12. It has been reported that the coenzyme B12 binding protein CarH, found in the carotenoid biosynthesis pathway of several thermostable bacteria, binds to the promoter region of DNA and suppresses transcription. To overcome the harmful effects of light-induced damage in the cells, CarH releases DNA in the presence of light and promotes transcription and synthesis of carotenoids, thereby working as a photoreceptor. CarH is able to achieve this by exploiting the photosensitive nature of the Co–C bond between the adenosyl moiety and the cobalt atom in the coenzyme B12 molecule. Extensive structural and spectroscopy studies provided a mechanistic understanding of the molecular basis of this unique light-sensitive reaction. Most studies on CarH have used the ortholog from the thermostable bacterium Thermus thermophilus, due to the ease with which it can be expressed and purified in high quantities. In this chapter we give an overview of this intriguing class of photoreceptors and report a step-by-step protocol for expression, purification and spectroscopy experiments (both static and time-resolved techniques) employed in our laboratory to study CarH from T. thermophilus. We hope the contents of this chapter will be of interest to the wider coenzyme B12 community and apprise them of the potential and possibilities of using coenzyme B12 as a light-sensing probe in a protein scaffold.

Original languageEnglish
Title of host publicationCoenzyme B Enzymes Part A
EditorsE. Neil G. Marsh
PublisherKluwer Academic/Human Sciences Press Inc.
Pages349-372
Number of pages24
ISBN (Print)9780323915915
DOIs
Publication statusPublished - Jan 2022

Publication series

NameMethods in Enzymology
Volume668
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Adenosylcobalamin
  • CarH
  • Cobalamin binding protein
  • Coenzyme B
  • Light-sensing
  • Photoreceptor
  • Protein purification
  • Transient absorbance spectroscopy

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