The urea carboxylase and allophanate hydrolase activities of urea amidolyase are functionally independent

Abstract

Urea amidolyase (UAL) is a multifunctional biotin-dependent enzyme that contributes to both bacterial and fungal pathogenicity by catalyzing the ATP-dependent cleavage of urea into ammonia and CO 2 . UAL is comprised of two enzymatic components: urea carboxylase (UC) and allophanate hydrolase (AH). These enzyme activities are encoded on separate but proximally related genes in prokaryotes while, in most fungi, they are encoded by a single gene that produces a fusion enzyme on a single polypeptide chain. It is unclear whether the UC and AH activities are connected through substrate channeling or other forms of direct communication. Here, we use multiple biochemical approaches to demonstrate that there is no substrate channeling or interdomain/intersubunit communication between UC and AH. Neither stable nor transient interactions can be detected between prokaryotic UC and AH and the catalytic efficiencies of UC and AH are independent of one another. Furthermore, an artificial fusion of UC and AH does not significantly alter the AH enzyme activity or catalytic efficiency. These results support the surprising functional independence of AH from UC in both the prokaryotic and fungal UAL enzymes and serve as an important reminder that the evolution of multifunctional enzymes through gene fusion events does not always correlate with enhanced catalytic function.

Structure and function of urea amidolyase

“…If there is no covalent linkage between the AH and UC domains, the latter is not strong enough to hold them to form a stable complex. This suggests that AH and UC encoded by separated genes probably do not form a KlUA-like complex, consistent with studies on a number of such AH and UC proteins [ 16 , 18 , 32 ].…”

“…Therefore, substrate channeling of allophanate within the same KlUA polypeptide or KlUA holo-enzyme is unlikely to play a role in the catalysis. A recent study on the Pseudomonas syringae UC and AH also ruled out the possibility that they form a transit complex to facilitate substrate channeling of allophanate during the catalysis [ 32 ].…”

Discovery of a widespread prokaryotic 5-oxoprolinase that was hiding in plain sight

“…However, the significance of this observation is unclear because inhibition was only observed when PxpB was expressed alone and not when PxpB and PxpC were co-expressed to form a complex, as our data show they normally would be. It has also recently been shown that certain fused pxpB and pxpC family genes, which cluster strongly with a urea carboxylase gene, are involved in urea catabolism and encode an enzyme with allophanate hydrolase activity (40,43). Allophanate hydrolase activity has only been demonstrated in a fused PxpBC homolog (43), but it is possible that other PxpB and PxpC proteins have dual roles in OP and urea metabolism.…”

“…It has also recently been shown that certain fused pxpB and pxpC family genes, which cluster strongly with a urea carboxylase gene, are involved in urea catabolism and encode an enzyme with allophanate hydrolase activity (40,43). Allophanate hydrolase activity has only been demonstrated in a fused PxpBC homolog (43), but it is possible that other PxpB and PxpC proteins have dual roles in OP and urea metabolism. Additionally, the prokaryote-type OPase genes of Ralstonia solanacearum were shown to be required for pathogenesis in tomato (44).…”

Allosteric regulation alters carrier domain translocation in pyruvate carboxylase

“…To ensure vector compatibility, the pET-28a vector encoding ΔBCΔBCCP RePC was modified to replace the pBR322 origin of replication with a pSC101 origin of replication. The gene encoding ΔBCΔBCCP RePC was re-cloned into vector pKLD66nCBP 31 . The T882A mutation was introduced into both ΔBC RePC and ΔBCΔBCCP RePC by whole plasmid mutagenesis using the Quikchange method.…”