The crystal structure of XdpB, the bacterial old yellow enzyme, in an FMN-free form

Abstract

Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal β-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress.

Crystal structure of ArOYE6 reveals a novel C‐terminal helical extension and mechanistic insights into the distinct class III OYEs from pathogenic fungi

“…Generally, the C‐terminal region of enzymatic proteins is involved in the regulatory mechanisms. Notably, at higher concentrations, the C‐terminal pentapeptide of a bacterial OYE homologue XdpB occupies the FMN‐binding site of other molecules, thus inactivating the enzyme by FMN displacement [45]. Therefore, we hypothesized that the uniquely extended C‐terminus of ArOYE6 might be responsible for its monomeric state as one such regulatory mechanism because other OYEs form dimers or tetramers.…”

Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: CtOYE and GsOYE

“…Finally, even though both enzymes belong to the classical OYE subfamily, which mostly encompasses non thermoresistant biocatalysts, they show a wide range of pH tolerance and solvent tolerance, comparable to that of thermophilic-like homologues. Reported melting points for classical OYEs range typically from 40 to 45 °C (e.g., Chr-OYE1 Tm = 40 °C (Pei et al 2016) and XdpB Tm = 39.8 °C (Zahradník et al 2018)). Surprisingly, CtOYE and GsOYE (with Tm of 53 °C and 67 °C, respectively) display significantly higher melting points (ΔTm = 15-25 °C), a property which renders these enzymes attractive for applications under demanding process conditions.…”