C-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and the resulting aglycones exert various bioactivities. Although the enzymes responsible for the reactions have been identified, their catalytic mechanisms and the generality of the reactions in nature remain to be explored. Here, we present the identification and structural basis for the activation of xenobiotic C-glycosides by heterocomplex C-deglycosylation enzymes from intestinal and soil bacteria. They are found to be metal-dependent enzymes exhibiting broad substrate specificity toward C-glycosides. X-ray crystallographic and cryo-electron microscopic analyses, as well as structure-based mutagenesis, reveal the structural details of these enzymes and the detailed catalytic mechanisms of their remarkable C-C bond cleavage reactions. Furthermore, bioinformatic and biochemical analyses suggest that the C-deglycosylation enzymes are widely distributed in the gut, soil, and marine bacteria.

LysR‐type transcription regulators (LTTRs) comprise one of the largest families of transcriptional regulators in bacteria. They are typically homo‐tetrameric proteins and interact with promoter DNA of ~ 50–60 bp. Earlier biochemical studies have suggested that LTTR binding to promoter DNA bends the DNA and, upon inducer binding, the bend angle of the DNA is reduced through a quaternary structure change of the tetrameric LTTR, leading to the activation of transcription. To date, crystal structures of full‐length LTTRs, DNA‐binding domains (DBD) with their target DNAs, and the regulatory domains with and without inducer molecules have been reported. However, these crystal structures have not provided direct evidence of the quaternary structure changes of LTTRs or of the molecular mechanism underlying these changes. Here, we report the first crystal structure of a full‐length LTTR, CbnR, in complex with its promoter DNA. The crystal structure showed that, in the absence of bound inducer molecules, the four DBDs of the tetrameric CbnR interact with the promoter DNA, bending the DNA by ~ 70°. Structural comparison between the DNA‐free and DNA‐bound forms demonstrates that the quaternary structure change of the tetrameric CbnR required for promoter region‐binding arises from relative orientation changes of the three domains in each subunit. The mechanism of the quaternary structure change caused by inducer binding is also discussed based on the present crystal structure, affinity analysis between CbnR and the promoter DNA, and earlier mutational studies on CbnR.Database: Atomic coordinates and structure factors for the full‐length Cupriavidus necator NH9 CbnR in complex with promoter DNA are available in the Protein Data Bank under the accession code 7D98.

Significance: Microbial degradation of C -glycosides is only reported from human intestinal bacteria. This study isolated a C -glycoside–catabolizing bacteria, Microbacterium sp. 5-2b, from soil and identified a C -glycoside–metabolizing enzyme. Surprisingly, while the metabolic pathway of C -glycoside in the strain was similar to that of an intestinal one, the enzyme named CarA catalyzing the initial step of metabolism was completely different from an enzyme identified from the intestinal microorganism; CarA identified as C -glycoside 3-oxidase for C-glycoside metabolism exhibited amino acid sequence similarity to pyranose oxidase family proteins. This study clarified the structure, function, and distribution of the enzyme involved in C -glycoside catabolism and provides insight into the biogeochemical circulation of C -glycosides in nature.

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.