Enzymatic divergence of GAPDH in Mycoplasma synoviae: Comparative insights from MS-H and TS4 strains

Mycoplasma synoviae causes respiratory infections and lameness in poultry, resulting in significant production losses. It carries two gene copies encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The M. synoviae strain MS-H, a live attenuated vaccine derived from the virulent isolate 86079/7NS (7NS), carries a distinct amino acid substitution per copy of gapdh. A field re-isolate of MS-H with increased pathogenicity, TS4, possesses a wild-type reversion in one of these copies, suggesting an association with virulence, but the functionality of each GAPDH variant is unclear. Here, a colorimetric assay was used to quantify the enzymatic activities of recombinant GAPDH variants VR, AK, and AR (each representing residues 185 and 306, respectively) expressed in Escherichia coli. The GAPDH variants were tested individually and in combination to represent MS-H (VR + AK), 7NS (AR), and TS4 (VR + AR), and the results were placed in contrast to those of crude cell lysates from 7NS, MS-H, and TS4 cultures. The AK and AR variants exhibited comparable enzymatic activity, whereas VR showed no detectable activity. Protein mixtures simulating MS-H and TS4 exhibited activity levels equivalent to AR and AK. The enzymatic activity in TS4 crude lysate was lower than in MS-H and 7NS. In-silico analysis revealed that the Ala185Val substitution in VR (which abolished its enzymatic activity) affects the NAD⁺ binding site, while the Arg306Lys in AK is close to the glyceraldehyde-3-phosphate binding site does not affect the NAD⁺ binding site. This study provides insights into how amino acid substitutions in GAPDH influence enzymatic function and highlights their complex role in M. synoviae pathogenesis.