Structural Superimposition

Abstact

Putrescine has long been considered the initiating substrate for aspartate semialdehyde-dependent bacterial spermidine biosynthesis. However, two recent studies identified a variant pathway in which agmatine serves as the preferred substrate. This variation appears to be common, but the mechanistic basis for substrate preference was unknown. Here, we investigate the aspartate semialdehyde-dependent dehydrogenases from Bacteroides fragilis and Clostridium leptum using steady-state and transient-state kinetic assays, mass spectrometry, thermal shift protein stability assays, X-ray crystallography, small-angle X-ray scattering, and computational modeling. Both enzymes exhibit dramatically enhanced catalytic efficiency with agmatine, demonstrating that these homologs function as carboxyaminopropylagmatine dehydrogenases rather than carboxyspermidine dehydrogenases. Agmatine enhances NADPH binding affinity by 34-fold, whereas putrescine has a modest effect and at concentrations less likely to be physiologically relevant. Apo crystal structures and small angle X-ray scattering analysis reveal substantial ligand-dependent conformational rearrangements involving both global domain and active-site loop motions. These structural transitions, together with thermal stability measurements and transient-state binding assays, support an induced-fit mechanism in which agmatine promotes conformational rearrangements that enhance NADPH-binding. Comparative sequence analysis of homologs and ligand docking lead us to propose an active-site recognition loop governing discrimination between agmatine, putrescine, and diaminopropane substrates. Together, these findings redefine the substrate specificity of the B. fragilis and C. leptum enzymes, establish a mechanistic model for agmatine-dependent catalysis in alternative spermidine biosynthesis, and provide a testable framework for predicting substrate specificity across this family of bacterial polyamine biosynthetic enzymes.