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Phytochromes are red-light photoreceptors first identified in plants, with homologs found in bacteria and fungi, that regulate a variety of critical physiological processes. They undergo a reversible photocycle between two distinct states: a red-light-absorbing Pr form and a far-red light-absorbing Pfr form. This Pr/Pfr photoconversion controls the activity of a C-terminal enzymatic domain, typically a histidine kinase (HK). However, the molecular mechanisms underlying light-induced regulation of HK activity in bacteria remain poorly understood, as only a few structures of unmodified bacterial phytochromes with HK activity are known. Recently, cryo-EM structures of a wild-type bacterial phytochrome with HK activity are solved that reveal homodimers in both the Pr and Pfr states, as well as a heterodimer with individual monomers in distinct Pr and Pfr states. Cryo-EM structures of a truncated version of the same phytochrome—lacking the HK domain—also show a homodimer in the Pfr state and a Pr/Pfr heterodimer. Here, we describe in detail how structural information is obtained from cryo-EM data on a full-length intact bacteriophytochrome, and how the cryo-EM structure can contribute to the understanding of the function of the phytochrome. In addition, we compare the cryo-EM structure to an unusual x-ray structure that is obtained from a fragmented full-length phytochrome crystallized in the Pr-state.
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This content will become publicly available on February 10, 2026
Structural and kinetic analysis of distinct active and inactive states of Burkholderia cenocepacia orotate phosphoribosyltransferase
Orotate phosphoribosyltransferase (OPRT) catalyzes the reaction that adds the pyrimidine base to the ribose in the penultimate step of the de novo biosynthesis of pyrimidine nucleotides. The OPRT structure consists of an obligate dimer, conserved throughout the phosphoribosyltransferase family. Here, we describe the structural characterization of Burkholderia cenocepacia OPRT (BcOPRT), both by X-ray crystallography and Cryo electron microscopy (Cryo-EM). While the known dimer is present in the structure of BcOPRT, a putative hexameric form was also observed by multiple methods. Analyses by chromatography, Cryo-EM, and kinetics indicate that both dimeric and hexameric forms of this enzyme are present together in solution. Comparison of the kinetics of the native protein and two variants, which were specifically designed to prevent hexamerization, reveal that only the hexameric form is enzymatically active. Collectively, these data suggest that BcOPRT may use oligomerization to control overall enzymatic activity, thus contributing to the local regulation of pyrimidine biosynthesis in this organism.
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- Award ID(s):
- 2149978
- PAR ID:
- 10572808
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Archives of Biochemistry and Biophysics
- Volume:
- 766
- Issue:
- C
- ISSN:
- 0003-9861
- Page Range / eLocation ID:
- 110332
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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