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.
Drexler et al. (Thu,) studied this question.