Abstract
Aspartate transcarbamoylase (ATCase) has been studied for decades and Escherichia coli ATCase is referred as a “textbook example” for both feedback regulation and cooperativity. However, several critical questions about the catalytic and regulatory mechanisms of E. coli ATCase remain unanswered, especially about its remote feedback regulation. Herein, we determined a structure of E. coli ATCase in which a key residue located (Arg167) at the entrance of the active site adopted an uncommon open conformation, representing the first wild-type apo-form E. coli ATCase holoenzyme that features this state. Based on the structure and our results of enzymatic characterization, as well as molecular dynamic simulations, we provide new insights into the feedback regulation of E. coli ATCase. We speculate that the binding of pyrimidines or purines would affect the hydrogen bond network at the interface of the catalytic and regulatory subunit, which would further influence the stability of the open conformation of Arg167 and the enzymatic activity of ATCase. Our results not only revealed the importance of the previously unappreciated open conformation of Arg167 in the active site, but also helped to provide rationalization for the mechanism of the remote feedback regulation of ATCase.
Highlights
Aspartate transcarbamoylase (E.C.2.1.3.2, aspartate carbamoyltransferase, ATCase) catalyzes the second step of the de novo biosynthesis of pyrimidines in most of organisms, which is the carbamoylation reaction between carbamoyl phosphate (CP) and L-aspartate (Asp), generating N-carbamoyl-L-aspartate (CA) and a single phosphate group (Pi) [1,2,3]
When the concentration of Asp increases to a certain level, most ATCase will be transformed to the R state to significantly enhance the catalyzing capacity of the carbamoylation reaction between CP and Asp, which is the mechanism of cooperativity [11,12]
The catalytic chain of ATCase can be divided into two domains: the CP domain and the Asp domain, and it is the closure of the two domains that trigger the T to R state transition of ATCase [5,13]
Summary
Aspartate transcarbamoylase (E.C.2.1.3.2, aspartate carbamoyltransferase, ATCase) catalyzes the second step of the de novo biosynthesis of pyrimidines in most of organisms, which is the carbamoylation reaction between carbamoyl phosphate (CP) and L-aspartate (Asp), generating N-carbamoyl-L-aspartate (CA) and a single phosphate group (Pi) [1,2,3]. The pyrimidine biosynthesis pathway of E. coli ATCase is physiologically controlled by altering the catalytic activity of ATCase through both cooperativity and feedback regulation [6,7], which has been referred to as a “textbook example” for decades. E. coli ATCase can adopt two states at quaternary level: a low activity and affinity T (tense) state and a high activity and affinity R (relax) state, where an equilibrium exists between the T and R state [8,9,10]. A low concentration of Asp cannot transform ATCase from the T to R state either. When the concentration of Asp increases to a certain level, most ATCase will be transformed to the R state to significantly enhance the catalyzing capacity of the carbamoylation reaction between CP and Asp, which is the mechanism of cooperativity [11,12]. Arg167 interacts with Asp and the intermediate product [16]
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