Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis

Abstract

In all organisms, carbamoylphosphate (CP) is a precursor common to the synthesis of arginine and pyrimidines. In Escherichia coli and most other Gram-negative bacteria, CP is produced by a single enzyme, carbamoylphosphate synthase (CPSase), encoded by the carAB operon. This particular situation poses a question of basic physiological interest: what are the metabolic controls coordinating the synthesis and distribution of this high-energy substance in view of the needs of both pathways? The study of the mechanisms has revealed unexpected moonlighting gene regulatory activities of enzymes and functional links between mechanisms as diverse as gene regulation and site-specific DNA recombination. At the level of enzyme production, various regulatory mechanisms were found to cooperate in a particularly intricate transcriptional control of a pair of tandem promoters. Transcription initiation is modulated by an interplay of several allosteric DNA-binding transcription factors using effector molecules from three different pathways (arginine, pyrimidines, purines), nucleoid-associated factors (NAPs), trigger enzymes (enzymes with a second unlinked gene regulatory function), DNA remodeling (bending and wrapping), UTP-dependent reiterative transcription initiation, and stringent control by the alarmone ppGpp. At the enzyme level, CPSase activity is tightly controlled by allosteric effectors originating from different pathways: an inhibitor (UMP) and two activators (ornithine and IMP) that antagonize the inhibitory effect of UMP. Furthermore, it is worth noticing that all reaction intermediates in the production of CP are extremely reactive and unstable, and protected by tunneling through a 96 Å long internal channel.

Keywords: Allosteric control; Arginine biosynthesis; Carbamoylphosphate synthase; DNA remodeling; Tandem promoters; Transcription regulation.

Fig. 1

Biosynthesis of arginine and pyrimidines. The names of structural genes encoding the enzymes catalyzing the different steps of de novo arginine and pyrimidine synthesis are indicated as follows: argA (N-acetylglutamate synthase), argB (N-acetylglutamate kinase), argC (N-acetylglutamylphosphate reductase), argD (N-acetylornithine transaminase), argE (N-acetylornithinase), argF and argI (ornithine transcarbamylase, two isoenzymes), argG (argininosuccinate synthetase), argH (argininosuccinase), carAB (carbamoylphosphate synthase), pyrBI (aspartate transcarbamylase with pyrB encoded catalytic and pyrI encoded regulatory subunit), pyrC (dihydroorotase), pyrD (dihydroorotate dehydrogenase), pyrE (orotate phophoribosyltransferase), pyrF (orotidine-5′-phosphate decarboxylase), pyrH (UMP kinase), pyrG (CTP-synthase)

Fig. 2

Sequence and outline of the E. coli carAB control region. P1 and P2 represent the tandem pair of promoters directing carAB transcription, with indication of their respective − 10 and − 35 promoter elements, and transcription initiation site(s) (arrows). Distances are indicated with respect to the start of P1 transcription. Boxed sequences represent binding sites for the various transcription factors and trigger enzymes modulating transcription initiation at the P1 and P2 promoters. The two ARG boxes constitute the binding site for hexameric arginine-bound ArgR. The PUR box is the target of dimeric guanine or hypoxanthine-bound PurR. The RUT box is the target of unliganded dimeric RutR. The IHF box is the binding site of heterodimeric integration host factor (IHF), a nucleoid-associated protein. PepA1 and PepA2 represent sites of tight contact with the hexameric trigger enzyme PepA as identified in DNase I footprinting. The approximately 230 bp long region that gets wrapped around PepA is underlined. Dam represents the GATC sequence that is methylated by deoxyadenosine methyltransferase, a reaction that is inhibited upon binding of PepA and/or IHF. Notice that the initiation codon for carA mRNA translation is TTG (in bold)

Fig. 3

Reaction scheme of the synthesis of carbamoylphosphate by E. coli CPSase using glutamine as nitrogen donor

Fig. 4

Ribbon presentation of the structure of heterodimeric (αβ) E. coli carbamoylphosphate synthase (pdb-1JDB) [133]. The small, carA encoded, glutamine amidotransferase subunit (magenta), and each domain of the large, carB encoded, catalytic subunit are indicated in different colors: carboxyphosphate synthetic component (green), oligomerization domain (yellow), carbamoylphosphate synthetic component (blue), allosteric domain (red). The figure was prepared with PyMOL