The widespread use of xenobiotics has driven the
rapid emergence of microbial degradation pathways. A prominent
example is enzymes involved in the catabolism of the herbicide
atrazine, which have evolved within the last few decades. Recently,
we provided evidence that the second enzyme of the atrazine
biodegradation pathway, hydroxyatrazine ethylaminohydrolase
(AtzB), has evolved from a progenitor enzyme of the
amidohydrolase superfamily (AtzB-CQNN) with guanine deaminase
(GuaD) activity. However, the catalytic efficiency for guanine
hydrolysis by AtzB-CQNN is several orders of magnitude lower
than that of prototypical GuaDs. In this study, we report a much
higher catalytic efficiency of AtzB-CQNN for the hydrolysis of the
guanine analogue N2,N2-dimethylguanine (kcat/KM ∼105−106 M−1s−1). This enzymatic activity has not been described up to now
and appears to be the native function of AtzB-CQNN, as well as that of several AtzB homologues termed NdmH. An active site
alanine scan of an NdmH enzyme allowed us to identify residues important for substrate binding and catalysis and to propose an
enzymatic reaction mechanism. The comparative characterization of NdmHs and canonical GuaDs revealed an extended substrate
scope and high evolvability of NdmH enzymes. A comprehensive computational evaluation, including conservation, covariance, and
flexibility studies, as well as conformational landscape reconstruction and correlation-based shortest path map analysis, showed that
this enhanced substrate promiscuity and evolvability of NdmHs compared with GuaDs are linked to a higher structural
heterogeneity of the active site, which facilitates their functional diversification to act on xenobiotics.