Ghazi Esfahani, B. | Search bioRxiv

bioRxiv2025

Mycoplasma penetrans Methionyl tRNA Synthetase is an Asymmetric Dimer fused to N-terminal Ancillary Domains

Diverse aminoacyl-tRNA synthetase (AARS) gene fusions are now recognized as a common mechanism for enhancing genetic diversity across all domains of life. The metS gene from Mycoplasma penetrans is a striking example of such an evolutionary mechanism because although M. penetrans has a condensed genome, the metS gene is nearly twice the size of a typical bacterial gene encoding methionyl tRNA synthetase (MetRS). We used cryo-EM to analyze the structure of the MpMetRS gene product to show that it is the product of three distinct enzyme domains: an N-terminal nucleotidyl transferase, a dimeric alanine-glyoxylate aminotransferase, and a MetRS. Only the N-terminal domains show two-fold symmetry, and the MetRS domain is only partially resolved. Modelling the full structure shows that a conformational change must occur to accommodate a tRNA-bound MetRS domain. A further rearrangement of the catalytic domains would also be necessary to bring the active sites adjacent to one another if this unique assembly of catalytic domains functions to channel substrates to MetRS.

biophysics↗

bioRxiv2024

Dimerization of assimilatory NADPH-dependent sulfite reductase reveals elements for diflavin reductase binding at a minimal interface

Escherichia coli NADPH-dependent assimilatory sulfite reductase (SiR) reduces sulfite by six electrons to make sulfide for incorporation into sulfur-containing biomolecules. SiR has two subunits: an NADPH, FMN, and FAD-binding diflavin flavoprotein and a siroheme/Fe4S4 cluster-containing hemoprotein. The molecular interactions that govern subunit binding have been unknown since the discovery of SiR over 50 years ago because SiR is flexible, thus has been intransigent for traditional high-resolution structural analysis. We used a combination of the chameleon(R) plunging system with a fluorinated lipid to overcome the challenges of preserving a flexible molecule to determine a 2.78 [A]-resolution cryo-EM structure of a minimal heterodimer complex. chameleon(R), combined with the fluorinated lipid, overcame persistent denaturation at the air-water interface. Using a previously characterized minimal heterodimer reduced the heterogeneity of a structurally heterogeneous complex to a level that could be analyzed using multi-conformer cryo-EM image analysis algorithms. Here, we report the first near-atomic resolution structure of the flavoprotein/hemoprotein complex, revealing how they interact in a minimal interface. Further, we determined the structural elements that discriminate between pairing a hemoprotein with a diflavin reductase, as in the E. coli homolog, or a ferredoxin partner, as in maize (Zea mays). Significance StatementSulfur is one of the essential building blocks of life. Sulfur exists in numerous redox states but only one can be incorporated into biomass - S2- (sulfide). In Escherichia coli, a protein enzyme called sulfite reductase reduces sulfite by six electrons to make sulfide. Typical electron transfer reactions move one or two electrons at a time. The sequential transfer of two electrons three times to complete the conversion of sulfite to sulfide (or nitrite to ammonia) is unique to sulfite or nitrite reductases. E. coli SiR is a two-protein complex composed of a diflavin reductase flavoprotein and an iron metalloenzyme hemoprotein. Until now, the molecular interactions that govern subunit interactions remained a mystery because the extreme flexibility of the flavoprotein subunit, which has challenged X-ray or cryo-EM analysis for over 30 years. In overcoming these challenges, we used a combination of rapid plunging with a high critical-micelle-concentration lipid alongside a biochemically minimized complex to determine the 2.78 [A]-resolution cryo-EM structure of a dimer between the flavoprotein and hemoprotein subunits.

molecular biology↗