Analysis of MHYT domain proteins and the regulation of enzymatic functions

The MHYT domain is named after its conserved amino acid motif. It has seven transmembrane domains, three of which have the conserved amino acid motif. The transmembrane domains are connected by arginine-rich cytoplasmic and histidine- and tyrosine-rich periplasmic loops. The residues of the conserved methionine, histidine and tyrosine amino acids are thought to bind copper, allowing them to sense diatomic gases. Although the first description of this domain is more than 20 years old, it was only this year that it could be shown experimentally that the MHYT domain is involved in gas perception [1, 2].

This study aims to investigate the heme binding to the MHYT domain and the influence of divalent cations on the modulation of activity of connected enzymatic domains. For this purpose, the genes coding for the MHYT domain proteins CdgB and NbdA were cloned for truncated and full-length variants and produced heterologously in presence of heme and the first precursor of heme, 5-aminolevulinic acid (ALA), in E. coli. In addition to the MHYT domain, both proteins have a diguanylate cyclase (DGC) and phosphodiesterase (PDE) domain. The solubilization of the full-length protein from E. coli membrane fractions using polymer nanodiscs enabled spectroscopic characterization of heme binding to the protein and activity assays. Absorption spectra of oxidized and reduced NbdA and CdgB revealed characteristic Soret bands for hemoproteins. The cytosolic NbdA variant showed PDE activity while interestingly the activity of the full-length protein was only shown when produced in E. coli Nissle 1917 in the presence of heme and iron ions. The cytosolic CdgB variant showed a possible DGC and PDE activity.

[1] Galperin MY, Gaidenko TA, Mulkidjanian AY, Nakano M, Price CW (2001) MHYT, a new integral membrane sensor domain. doi: 10.1111/j.1574-6968.2001.tb10919.x

[2] Durante-Rodríguez G, Francisco-Polanco S de, García JL, Díaz E (2024) Characterization of a MHYT domain-coupled transcriptional regulator that responds to carbon monoxide. doi: 10.1093/nar/gkae575