Comparative structural analysis provides new insights into the function of R2‐like ligand‐binding oxidase

Abstract

R2‐like ligand‐binding oxidase (R2lox) is a ferritin‐like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross‐link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry‐based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.

A novel alkane monooxygenase evolved from a broken piece of ribonucleotide reductase in Geobacillus kaustophilus HTA426 isolated from Mariana Trench

“…No such activity was detected for either of the enzymes. This indicates that fatty aldehydes are not R2lox substrates (Mak et al 2020 ; Diamanti et al 2022 ).…”

“…The crystal structure of Gk R2loxI, 4HR0_A, has already been reported, and is the first R2lox homolog in the genus Geobacillus (Griese et al 2013 ). Interestingly, the recombinant Gk R2loxI produced in E. coli was co-purified with C16 or C18 hydroxy fatty acids (Griese et al 2013 ; Diamanti et al 2022 ). Based on this information, we hypothesized that Gk R2loxI (GK2771) functions as a novel alkane hydroxylase in the alkane degradation system.…”

Adaptive introgression reveals the genetic basis of a sexually selected syndrome in wall lizards

“…The peaks of the absorption spectra were compared to known absorption spectra of carotenoids. Extracted pigments from budgerigar feathers (using acidified pyridine) and from lizard skin (using acetone) were transferred to the Swedish Metabolomics Center for LC-HRMS using ESI following procedures previously described in ( 65 ). LC-HRMS analysis was performed on a 1290 Infinity system from Agilent Technologies (Waldbronn, Germany), with an Agilent 6550 QTOF mass spectrometer for MS detection.…”

Site-Differentiated MnIIFeII Complex Reproducing the Selective Assembly of Biological Heterobimetallic Mn/Fe Cofactors

“…Heterobimetallic cofactors have been discovered in a number of proteins responsible for life-sustaining chemical transformations, including the heme a 3 /Cu B site in cytochrome c oxidase, [NiFe] hydrogenase, Cu/Zn superoxide dismutase, and the Mn/Fe or Zn/Fe sites in purple acid phosphatases . Mn/Fe cofactors have also been characterized in class Ic ribonucleotide reductases (RNRIc), − R2-like ligand-binding oxidases (R2lox), − and, most recently, in the chlamydia protein associated with death domains (CADD) . Since their discovery, heterobimetallic cofactors have piqued the interest of chemists and biologists alike.…”

“…The mechanism of heterobimetallic Mn II Fe II cofactor assembly in vivo has been the subject of intense scrutiny. Work by Högbom et al, ,,− Shafaat et al, − Stubbe et al, − and Bollinger et al − suggests that favorable Mn II Fe II assembly is likely driven by a combination of metal availability and differential binding affinity. While in vivo studies have provided valuable insights into the selective formation of Mn II Fe II cofactors, the complexity of the protein matrix has prevented the identification of specific structural features responsible for this process.…”