# Stringent Response in Mycobacteria: From Biology to Therapeutic Potential
## 2 Metabolism of (p)ppGpp in Mycobacterium  
The genes encoding the enzymes for (p)ppGpp metabolism have been found in all sequenced bacterial genomes--except the phyla Chlamydia, Verrucomicrobia, Planctomycetes, and a few obligate intracellular symbiotic bacterial species--which makes stringent response a nearly ubiquitous phenomenon in bacteria [43]. In Gram-negative bacteria, the stringent response is governed by two enzymes--RelA and SpoT. RelA, encoded by the _relA_ gene, is a monofunctional synthetase responsible for the synthesis of (p)ppGpp. On the other hand, the bifunctional SpoT, encoded by the _spoT_ gene, acts primarily as a hydrolase responsible for the degradation of (p)ppGpp. SpoT can also synthesize (p)ppGpp in response to stress, which does not activate RelA. In mycobacteria, the alarmones are synthesized and degraded by a dual-function enzyme, Rel [44]. It is believed that RelA and SpoT have evolved from the same ancestral Rel protein, and the hydrolase domain has been inactive in RelA. Thus, RelA, SpoT and Rel proteins have similar domain architecture and have been characterized as the RelA Spo Homology (RSH) superfamily of proteins [43].  
Both pathogenic and non-pathogenic mycobacterial genomes have a _rel_ gene, which encodes bifunctional Rel protein [43]. In _M. tuberculosis_, the gene _rv2583c_ encodes this bifunctional Rel, which is a 790 amino acid long multidomain protein, comprising catalytic N-terminal domain (1-394 aa) and a regulatory C-terminal domain (395-790 aa). The N-terminal domain harbors both the hydrolase activity (1-181 aa) and the synthetase activity (87-394 aa) [43; 45; 46]. Amino acid residues 87-181 are shared between the hydrolase and synthetase activities of the N-terminal domain and form a three-helix bundle. Both the enzymatic activities require Mn\({}^{2+}\) or Mg\({}^{2+}\) cations as co-factors [27; 44; 46] (Figure 1A).  
Several bacteria including mycobacteria also encode homologs of RSH proteins, which are smaller in length. These proteins are usually single-domain proteins and possess either synthetase or hydrolase activity. Hence, they are called small alarmone synthetases (SASs) or small alarmone hydrolases (SAH) [47; 48; 49; 50]. The genomes of both _M. tuberculosis_ and _M. smegmatis_ contain one copy of SAS [30; 47; 51; 52] (Figure 1A). In _M. tuberculosis_, the gene _rv1366_ was predicted to encode a potential SAS. However, the Rv1366 protein was shown to be catalytically inactive as it could not synthesize (p)ppGpp [51]. The saprophy _M. smegmatis_ encodes RelZ, a SAS protein, which can synthesize the third alarmone, pGpp, unlike its _M. tuberculosis_ ortholog Rv1366 (Figure 1). RelZ protein also possesses N-terminal RNase HII domain, which removes RNA-DNA hybrids generated during DNA replication [52]. The presence of both RNase HII and pGpp synthetase domains is needed for the RelZ catalytic activity, as individual domains have been shown to be enzymatically inactive. The catalytic inactivation of one domain does not impair the enzymatic activity of the other domain [52]. RelZ prefers GMP as substrate, unlike RelMtb, which prefers GDP/GTP as the substrate. Although no SAH has been identified in mycobacteria, RelMsm cleaves pGpp to GMP and pyrophosphate [30] (Figure 1B).