Nicholas Schneider (Marquette University)| Lambros Tassoulas (University of Minnesota)| Danyun Zeng (Marquette University)| Amanda Laseke (Marquette University)| Nicholas Reiter (Marquette University)| Lawrence Wackett (University of Minnesota)| Martin St. Maurice (Marquette University)
Urea amidolyase (UAL) is a biotin-dependent enzyme composed of urea carboxylase (UC) and allophanate hydrolase (AH). UAL catalyzes the cleavage of urea into ammonia and CO2 through an ATP and HCO3- -dependent reaction. Curiously, many bacteria also have the enzyme urease which also can degrade urea, but through an ATP-independent reaction. An explanation for this conundrum has remained elusive, but we propose a solution. It has recently been reported that the expression of bacterial UC can be regulated by a guanidine-sensitive riboswitch. Additionally, bacterial UC has a catalytic efficiency of ATP cleavage that is ~40-fold higher in the presence of guanidine compared to urea. This suggests that guanidine is a preferred substrate for UC and that guanidine may be degraded into ammonia and CO2 by a similar pathway to urea. Here, we confirm that the ATP cleavage is accelerated in the presence of guanidine compared to urea. However, we did not observe the complete degradation of guanidine to ammonia and CO2 through the combined actions of UC and AH. The genes of UC and AH are often clustered with two genes of unknown function, annotated as urea carboxylase-associated family proteins. We reveal that these genes encode the subunits of a heteromeric carboxyguanidine deiminase (CgdAB), which is required for the complete degradation of guanidine to ammonia and CO2 in the presence of UC and AH. We propose that the guanidine degradation pathway includes carboxylation of guanidine to form carboxyguanidine by UC, deimination of carboxyguanidine to form allophanate by CgdAB, and hydrolysis of allophanate to ammonia and CO2 by AH. Interestingly, we reveal that the fungal UAL enzymes of Candida albicans and Saccharomyces cerevisiae are unable to degrade guanidine in the presence of CgdAB and do not use guanidine as a substrate. We show that 75% of bacterial UC genes do colocalize with a cgdAB gene, and all fungal UAL genes do not. A subtle enzymatic difference of the fungal UALs and the bacterial UCs is the presence of an Asp/Asn residue mutation in the carboxyltransferase active site. Additionally, most of the 25% of bacterial UC enzymes whose genes do not colocalize with cgdAB also contain an Asn, consistent with the variation observed in fungal UALs. The exclusive presence of CgdAB in bacteria suggests that a mechanism for metabolic guanidine degradation has independently evolved in the bacteria relative to the fungi.