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Comparison of the external aldimine vs. internal aldimine in GabR. External aldimine Schiff bases (magenta and cyan), internal aldimine Schiff base with K312 (gray), and Y281 are shown in stick form. Labels and carbon atoms for internal aldimine (PDB code ID 4N0B) are in black and gray, respectively. Labels and carbon atoms for the Eb/O-PLP-GABA complex are in magenta. Labels and carbon atoms for the Eb/O-PLP-AFPA complex are in cyan.
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Significance
Regulator of the gabTD operon and its own gene (GabR) is an intriguing case of molecular evolution, displaying the evolutionary lineage between a pyridoxal-5′-phosphate (PLP)-dependent aminotransferase and a regulation domain of a transcription regulator. Here, PLP’s native function is not a catalytic coenzyme, but an effector of trans...
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Context 1
... GabRmediated regulation are illustrated. The effectors for GabR-mediated transcription activation are shown in dashed boxes. The GABA-AT reaction in a "Ping-Pong" mechanism is shown in the solid box. catalytic residue during catalytic reactions, such as in the first half of the "Ping-Pong" transamination (6) (Scheme S1). The conserved K312 (9) (Fig. 4) was previously proven to be essential for GabR's function, although the catalytic capacity to facilitate transamination has never been detected for GabR (8). In the Eb/ O-PLP-GABA complex structure ( Fig. 2A), Y281 is observed to block K312 from accessing the PLP-GABA Schiff base, preventing reformation of the internal aldimine as ...
Context 2
... would deprotonate the carbon adjacent to the nitrogen atom of the GABA-PLP adduct (19,22) (Scheme S1). As a result, GabR is unlikely to catalyze the first half of the transamination reaction shown in Scheme 1. As we previously reported (9), in the absence of GABA, Y281 is found in a different orientation, allowing internal aldimine formation (Fig. 4). The observed obstruction by Y281 supersedes the previous speculation that GabR's inability to stabilize the external aldimine might be the reason for the lack of observed transaminase activity ...
Context 3
... (Fig. 5A). To validate the existence of the fluorine atom, extra rounds of refinement were done after deletion of the fluorine atom; the omit map difference electron density (Fo-Fc) strongly supports the presence of the fluorine atom in the complex. Residue Y281 is observed to be in the same position as in the Eb/O-PLP-GABA complex structure (Fig. 4), preventing the access of K312 to the PLP-AFPA Schiff base, which could otherwise potentially push the reaction further along the proposed inactivation pathway (Scheme ...
Citations
... These characteristics suggest that MpaR is a member of the MocR subfamily within the GntR family of bacterial transcription factors (56). Although members of the MocR subfamily contain a domain with homology to PLPdependent aminotransferases, studies have indicated that this domain is not enzymati cally active and that in these proteins, binding of PLP and/or related compounds instead serves as a modulator of transcription factor function (57)(58)(59)(60). In Bacillus clausii PdxR, for example, it has been shown that the binding of PLP to the C-terminal domain can affect the conformation of the N-terminal domain, thereby changing the protein's affinity for distinct DNA-binding motifs (61). ...
Pseudomonas aeruginosa is a common, biofilm-forming pathogen that exhibits complex pathways of redox metabolism. It produces four different types of terminal oxidases for aerobic respiration, and for one of these, the cbb 3 -type terminal oxidases, it has the capacity to produce at least 16 isoforms encoded by partially redundant operons. It also produces small-molecule virulence factors that interact with the respiratory chain, including the poison cyanide. Previous studies had indicated a role for cyanide in activating expression of an “orphan” terminal oxidase subunit gene called ccoN4 and that the product contributes to P. aeruginosa cyanide resistance, fitness in biofilms, and virulence, but the mechanisms underlying this process had not been elucidated. Here, we show that the regulatory protein MpaR, which is predicted to be a pyridoxal phosphate-binding transcription factor and is encoded just upstream of ccoN4 , controls ccoN4 expression in response to endogenous cyanide. Paradoxically, we find that cyanide production is required to support CcoN4’s contribution to respiration in biofilms. We identify a palindromic motif required for cyanide- and MpaR-dependent expression of ccoN4 and co-expressed adjacent loci. We also characterize the regulatory logic of this region of the chromosome. Finally, we identify residues in the putative cofactor-binding pocket of MpaR, which are required for ccoN4 expression. Together, our findings illustrate a novel scenario in which the respiratory toxin cyanide acts as a signal to control gene expression in a bacterium that produces the compound endogenously.
IMPORTANCE
Cyanide is an inhibitor of heme-copper oxidases, which are required for aerobic respiration in all eukaryotes and many prokaryotes. This fast-acting poison can arise from diverse sources, but mechanisms by which bacteria sense it are poorly understood. We investigated the regulatory response to cyanide in the pathogenic bacterium Pseudomonas aeruginosa , which produces cyanide as a virulence factor. Although P. aeruginosa has the capacity to produce a cyanide-resistant oxidase, it relies primarily on heme-copper oxidases and even makes additional heme-copper oxidase proteins specifically under cyanide-producing conditions. We found that the protein MpaR controls expression of cyanide-inducible genes in P. aeruginosa and elucidated the molecular details of this regulation. MpaR contains a DNA-binding domain and a domain predicted to bind pyridoxal phosphate (vitamin B6), a compound that is known to react spontaneously with cyanide. These observations provide insight into the understudied phenomenon of cyanide-dependent regulation of gene expression in bacteria.
... To date, only a few MocR-TFs have been characterized molecularly, most of which are involved in the metabolism of nitrogen-containing compounds. GabR, for example, regulates transcription of γ-aminobutyric acid (GABA) aminotransferase (GabT) gene and promotes the catabolism of glutamate from GABA in Bacillus subtilis (Al-Zyoud et al., 2015;Edayathumangalam et al., 2013;Wu et al., 2017). PdxR, a transcriptional regulator that activates transcription of the pdxST genes encoding Pyridoxal 5 0 -phosphate (PLP) synthase in Bacillus clausii (Qaidi et al., 2013;Tramonti et al., 2015). ...
... However, no reports on its aspartate aminotransferase activities has been found ever. They often involves in controlling the metabolism of organic nitrogen compounds (Belitsky, 2014;Tramonti et al., 2016;Wu et al., 2017). Previous studies showed that most MocR-TFs encoding genes and their target genes are located together with divergent orientations. ...
... MocR-TFs binds this region to represses its own expression, but activates the expression of the structure genes. For example, GabR, regulating GABA metabolism in Bacillus subtilis, is an autorepressor and transcriptional activator of gabR and gabT, respectively, which allows the bacterium to use GABA as nitrogen and carbon sources (Okuda et al., 2014;Wu et al., 2017). PdxR, involved in the regulation of pyridoxal phosphate synthesis in Bacillus clausii, activates the transcription of pdxST genes encoding PLP synthase while represses its own expression (Tramonti et al., 2015). ...
Microbial ammonia oxidation is vital to the nitrogen cycle. A biological process, called Dirammox (direct ammonia oxidation, NH3→NH2OH→N2), has been recently identified in Alcaligenes ammonioxydans and A. faecalis. However, its transcriptional regulatory mechanism has not yet been fully elucidated. The present study characterized a new MocR‐like transcription factor DnfR that is involved in the Dirammox process in A. faecalis strain JQ135. The entire dnf cluster was composed of ten genes and transcribed as five transcriptional units, i.e., dnfIH, dnfR, dnfG, dnfABCDE and dnfF. DnfR activates the transcription of dnfIH, dnfG, and dnfABCDE genes, and represses its own transcription. The intact 1,506‐bp dnfR gene was required for activation of Dirammox. Electrophoretic mobility shift assays and DNase I footprinting analyses showed that DnfR has one binding site in the dnfH‐dnfR intergenic region and two binding sites in the dnfG‐dnfA intergenic region. Three binding sites of DnfR shared a 6‐bp repeated conserved sequence 5’‐GGTCTG‐N17‐GGTCTG‐3’ which was essential for the transcription of downstream target genes. Cysteine and glutamate act as possible effectors of DnfR to activate the transcription of transcriptional units of dnfG and dnfABCDE, respectively. This study provided new insights in the transcriptional regulation mechanism of Dirammox by DnfR in A. faecalis JQ135. This article is protected by copyright. All rights reserved.
... In the absence of GABA, GabR is an autorepressor (Belitsky and Sonenshein 2002;Edayathumangalam et al., 2013). GabR is an interesting transcriptional regulator that contains an aminotransferase domain and has been shown to bind both pyridoxal-5′phosphate (PLP) and GABA, which react to form an aldimine that activates GabR-mediated transcription of the P GabTD promoter via a dimeric GabR protein complex (Edayathumangalam et al., 2013;Okuda et al., 2015;Wu et al., 2017). The P GabTD promoter has been well-studied and was proposed to have three putative DNA binding sites for GabR, comprised of two 6-nt direct repeats that have an inverted repeat between them (Edayathumangalam et al., 2013;Nardella et al., 2020). ...
Engineered probiotic bacteria have been proposed as a next-generation strategy for noninvasively detecting biomarkers in the gastrointestinal tract and interrogating the gut-brain axis. A major challenge impeding the implementation of this strategy has been the difficulty to engineer the necessary whole-cell biosensors. Creation of transcription factor-based biosensors in a clinically-relevant strain often requires significant tuning of the genetic parts and gene expression to achieve the dynamic range and sensitivity required. Here, we propose an approach to efficiently engineer transcription-factor based metabolite biosensors that uses a design prototyping construct to quickly assay the gene expression design space and identify an optimal genetic design. We demonstrate this approach using the probiotic bacterium Escherichia coli Nissle 1917 (EcN) and two neuroactive gut metabolites: the neurotransmitter gamma-aminobutyric acid (GABA) and the short-chain fatty acid propionate. The EcN propionate sensor, utilizing the PrpR transcriptional activator from E. coli, has a large 59-fold dynamic range and >500-fold increased sensitivity that matches biologically-relevant concentrations. Our EcN GABA biosensor uses the GabR transcriptional repressor from Bacillus subtilis and a synthetic GabR-regulated promoter created in this study. This work reports the first known synthetic microbial whole-cell biosensor for GABA, which has an observed 138-fold activation in EcN at biologically-relevant concentrations. Using this rapid design prototyping approach, we engineer highly functional biosensors for specified in vivo metabolite concentrations that achieve a large dynamic range and high output promoter activity upon activation. This strategy may be broadly useful for accelerating the engineering of metabolite biosensors for living diagnostics and therapeutics.
... MocR-like transcriptional regulator DnfR positively regulates dnf(ABC) af expression in strain JQ135. In the dnf cluster, a 1,506-bp gene (AFA_18585, designated dnfR) (Fig. 5A) was annotated as a putative MocR-family transcriptional regulator (24,25). DnfR contains a GntR-family transcriptional regulator domain (Gln 20 to Ser 85 ) and a pyridoxal phosphate (PLP)-dependent aminotransferase domain (Ala 140 to Leu 490 ). ...
Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (direct ammonia oxidation, NH3 !NH2OH!N2) has been identified in Alcaligenes ammonioxydans. However, whether Dirammox is present in other microbes, as well as its genetic regulation, remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N2 via NH2OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N2O and trace amounts of N2, with nitrite as the sole nitrogen source. Deletion of the nirK and nosZ genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 59-TGGTCTGT-39 in the promoter region. A bioinformatic survey showed that homologs of dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR.
... Depending on the type and fold of the C-terminal domain, GntR-type transcription factors can be divided into several sub-families (Rigali et al., 2002;Jain, 2015); one of them is formed by MocR/GabR-type proteins (Rossbach et al., 1994;Bramucci et al., 2011;Suvorova and Rodionov, 2016;Tramonti et al., 2018;Pascarella, 2019). The genetically, biochemically, and structurally best characterized member of this sub-family is the GabR protein from Bacillus subtilis, a regulatory protein involved in the utilization of γ-amino-butyric acid (GABA) as a nitrogen source (Belitsky and Sonenshein, 2002;Belitsky, 2004;Edayathumangalam et al., 2013;Wu et al., 2017;Nardella et al., 2020). ...
... The C-terminal effector-binding and oligomerization domains of MocR/GabR-type proteins resemble in their fold that of aminotransferases of type I, enzymes that depend on the cofactor pyridoxal-5 -phosphate (PLP, vitamin B6) for their activity (Percudani and Peracchi, 2003;Belitsky, 2004;Suvorova and Rodionov, 2016;Tramonti et al., 2018;Richts et al., 2019). However, the aminotransferase domain (ATD) of these regulatory proteins does not possess enzymatic activity; instead it is used as a sensory domain to affect DNA-binding in response to environmental or cellular cues (Percudani and Peracchi, 2003;Okuda et al., 2015b;Wu et al., 2017;Tramonti et al., 2018). ...
... In other MocR/GabR-type regulators, the covalently bound PLP interacts chemically with system-specific low molecular mass inducer molecules (Edayathumangalam et al., 2013;Okuda et al., 2015b;Tramonti et al., 2018). Stemming from this interaction, internal and external aldimines are formed, thereby triggering a conformational change that affects the DNA-binding properties of the transcription factor (Edayathumangalam et al., 2013;Okuda et al., 2015b;Wu et al., 2017;Tramonti et al., 2018;Frezzini et al., 2020). The term internal aldimine refers to a PLP molecule covalently bound via a Schiff-base to the side chain of a lysine residue. ...
The compatible solutes ectoine and 5-hydroxyectoine are widely synthesized by bacteria as osmostress protectants. These nitrogen-rich tetrahydropyrimidines can also be exploited as nutrients by microorganisms. Many ectoine/5-hydroxyectoine catabolic gene clusters are associated with a regulatory gene ( enuR : ectoine nutrient utilization regulator) encoding a repressor protein belonging to the MocR/GabR sub-family of GntR-type transcription factors. Focusing on EnuR from the marine bacterium Ruegeria pomeroyi , we show that the dimerization of EnuR is mediated by its aminotransferase domain. This domain can fold independently from its amino-terminal DNA reading head and can incorporate pyridoxal-5′-phosphate (PLP) as cofactor. The covalent attachment of PLP to residue Lys302 of EnuR was proven by mass-spectrometry. PLP interacts with system-specific, ectoine and 5-hydroxyectoine-derived inducers: alpha-acetyldiaminobutyric acid (alpha-ADABA), and hydroxy-alpha-acetyldiaminobutyric acid (hydroxy-alpha-ADABA), respectively. These inducers are generated in cells actively growing with ectoines as sole carbon and nitrogen sources, by the EutD hydrolase and targeted metabolic analysis allowed their detection. EnuR binds these effector molecules with affinities in the low micro-molar range. Studies addressing the evolutionary conservation of EnuR, modelling of the EnuR structure, and docking experiments with the inducers provide an initial view into the cofactor and effector binding cavity. In this cavity, the two high-affinity inducers for EnuR, alpha-ADABA and hydroxy-alpha-ADABA, are positioned such that their respective primary nitrogen group can chemically interact with PLP. Purified EnuR bound with micro-molar affinity to a 48 base pair DNA fragment containing the sigma-70 type substrate-inducible promoter for the ectoine/5-hydroxyectoine importer and catabolic gene cluster. Consistent with the function of EnuR as a repressor, the core elements of the promoter overlap with two predicted EnuR operators. Our data lend themselves to a straightforward regulatory model for the initial encounter of EnuR-possessing ectoine/5-hydroxyectoine consumers with environmental ectoines and for the situation when the external supply of these compounds has been exhausted by catabolism.
... Pseudoenzyme transcription factors are also found in the superfamily of proteins that bind pyridoxal 5 0 -phosphate (PLP) as a cofactor in the active site. The MocR/GabR subfamily includes GabR and several other transcription factors that possess a Cterminal aminotransferase-like domain that binds PLP but is catalytically inactive and an N-terminal winged-helix domain that binds DNA [49][50][51][52][53][54][55][56]. It is likely that the DNA-binding domain was added to an ancestral aminotransferase enzyme to result in an intermediate moonlighting protein before loss of catalytic activity to result in the pseudoenzyme transcription factor. ...
... Despite the fact that many bacteria possess the characteristic genes involved in GABA metabolism, their regulation has so far only been studied in a few species such as Bacillus subtilis, Bacillus thuringiensis, or Escherichia coli. The best-studied regulator is probably GabR of B. subtilis (designated here GabR Bs ), which belongs to the MocR/GabR subfamily of the GntR family of transcriptional regulators (Edayathumangalam et al., 2013;Wu et al., 2017). GabR Bs consists of an N-terminal helixturn-helix (HTH) domain and a C-terminal aminotransferase domain and activates its target genes gabTD in the presence of GABA and PLP (Belitsky, 2004). ...
... GabR might bind to its target DNA both in the apo-state and in the ligandbound state, but requires binding of GABA or another effector metabolite to trigger a conformational change that is necessary to activate transcription of gabTDP. Such a situation was found e.g., for B. subtilis GabR Bs , which binds to its target promoter independent of GABA, but transcription activation of the target genes requires binding of GABA and PLP (Wu et al., 2017). However, GabR Bs belongs to a different protein family than GabR of C. glutamicum and also PLP had no influence on GabR binding to DNA, neither alone nor in combination with GABA (Supplementary Figure 9). ...
γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid mainly formed by decarboxylation of L-glutamate and is widespread in nature from microorganisms to plants and animals. In this study, we analyzed the regulation of GABA utilization by the Gram-positive soil bacterium Corynebacterium glutamicum, which serves as model organism of the phylum Actinobacteria. We show that GABA usage is subject to both specific and global regulatory mechanisms. Transcriptomics revealed that the gabTDP genes encoding GABA transaminase, succinate semialdehyde dehydrogenase, and GABA permease, respectively, were highly induced in GABA-grown cells compared to glucose-grown cells. Expression of the gabTDP genes was dependent on GABA and the PucR-type transcriptional regulator GabR, which is encoded divergently to gabT. A ΔgabR mutant failed to grow with GABA, but not with glucose. Growth of the mutant on GABA was restored by plasmid-based expression of gabR or of gabTDP, indicating that no further genes are specifically required for GABA utilization. Purified GabR (calculated mass 55.75 kDa) formed an octamer with an apparent mass of 420 kDa and bound to two inverted repeats in the gabR-gabT intergenic region. Glucose, gluconate, and myo-inositol caused reduced expression of gabTDP, presumably via the cAMP-dependent global regulator GlxR, for which a binding site is present downstream of the gabT transcriptional start site. C. glutamicum was able to grow with GABA as sole carbon and nitrogen source. Ammonium and, to a lesser extent, urea inhibited growth on GABA, whereas L-glutamine stimulated it. Possible mechanisms for these effects are discussed.
... MocR/GabR-type regulators typically consist of a N-terminal winged-helix-turn-helix DNA-reading head connected via a long and highly flexible linker to a C-terminal aminotransferase domain of fold I (Edayathumangalam et al. 2013;Tramonti et al. 2018). The aminotransferase domain does not possess enzymatic function; instead the chemistry of a covalently bound PLP in a reaction with a systems-specific low-molecular-weight effector molecule triggers a conformational change affecting DNA binding (Edayathumangalam et al. 2013;Frezzini et al. 2020;Tramonti et al. 2018;Wu et al. 2017). ...
Ectoine and its derivative 5-hydroxyectoine are compatible solutes and chemical chaperones widely synthesized by Bacteria and some Archaea as cytoprotectants during osmotic stress and high- or low-growth temperature extremes. The function-preserving attributes of ectoines led to numerous biotechnological and biomedical applications and fostered the development of an industrial scale production process. Synthesis of ectoines requires the expenditure of considerable energetic and biosynthetic resources. Hence, microorganisms have developed ways to exploit ectoines as nutrients when they are no longer needed as stress protectants. Here, we summarize our current knowledge on the phylogenomic distribution of ectoine producing and consuming microorganisms. We emphasize the structural enzymology of the pathways underlying ectoine biosynthesis and consumption, an understanding that has been achieved only recently. The synthesis and degradation pathways critically differ in the isomeric form of the key metabolite N-acetyldiaminobutyric acid (ADABA). γ-ADABA serves as preferred substrate for the ectoine synthase, while the α-ADABA isomer is produced by the ectoine hydrolase as an intermediate in catabolism. It can serve as internal inducer for the genetic control of ectoine catabolic genes via the GabR/MocR-type regulator EnuR. Our review highlights the importance of structural enzymology to inspire the mechanistic understanding of metabolic networks at the biological scale.
... Each subunit of GabR consists of an N-terminal winged helix-turn-helix (wHTH) DNAbinding domain and a C-terminal effector-binding/oligomerization (eb/o) domain (Figure 1b) homologous to highly conserved Type-I aminotransferases, such as GABA-AT and aspartate aminotransferase (Asp-AT). [2][3][4] In 2015, Wu et al. reported that GabR facilitates a "partial" aminotransferase-like reaction involving GABA and the cofactor pyridoxal-5 0 -phosphate (PLP), forming an external aldimine. However, GabR is incapable of completing the "Ping-Pong" transamination reaction due to conformational restraints imposed by Tyr 281. 4 As a result, GabR functions solely as a PLP-dependent transcriptional regulator, responding to the concentration of GABA but without catalytic capacity. 4 The current GabR-DNA binding model suggests that GabR binds as a homodimer to regulate transcription. ...
... However, GabR is incapable of completing the "Ping-Pong" transamination reaction due to conformational restraints imposed by Tyr 281. 4 As a result, GabR functions solely as a PLP-dependent transcriptional regulator, responding to the concentration of GABA but without catalytic capacity. 4 The current GabR-DNA binding model suggests that GabR binds as a homodimer to regulate transcription. GabR specifically recognizes GABA, which induces protein conformational changes upon binding. ...
... However, GabR is incapable of completing the "Ping-Pong" transamination reaction due to conformational restraints imposed by Tyr 281. 4 As a result, GabR functions solely as a PLP-dependent transcriptional regulator, responding to the concentration of GABA but without catalytic capacity. 4 The current GabR-DNA binding model suggests that GabR binds as a homodimer to regulate transcription. GabR specifically recognizes GABA, which induces protein conformational changes upon binding. ...
Addressing molecular recognition in the context of evolution requires pursuing new molecular targets to enable the development of agonists or antagonists with new mechanisms of action. Disruption of transcriptional regulation through targeting transcription factors that regulate the expression of key enzymes in bacterial metabolism may provide a promising method for controlling the bacterial metabolic pathways. To this end, we have selectively targeted a bacterial transcription regulator through the design and synthesis of a series of γ‐aminobutyric acid (GABA) derivatives, including (S)‐4‐amino‐5‐phenoxypentanoate (4‐phenoxymethyl‐GABA), which are based on docking insights gained from a previously‐solved crystal structure of GabR from Bacillus subtilis. This target was selected because GabR strictly controls GABA metabolism by regulating the transcription of the gabT/D operon. These GabR transcription modulators are selective for the bacterial transcription factor GabR and are unable to bind to structural homologs of GabR due to distinct steric constraints. We have obtained a crystal structure of 4‐phenoxymethyl‐GABA bound as an external aldimine with PLP in the effector binding site of GabR, which suggests that this compound is capable of binding and reacting in the same manner as the native effector ligand. Inhibition assays demonstrate high selectivity of 4‐phenoxymethyl‐GABA for bacterial GabR versus several selected eukaryotic enzymes. Single‐molecule fluorescence resonance energy transfer (smFRET) experiments reveal a ligand‐induced DNA distortion that is very similar to that of the native effector GABA, suggesting that the compound functions as a potential selective agonist of GabR.
... Pseudoenzyme transcription factors are also found in the superfamily of proteins that bind pyridoxal 5 0 -phosphate (PLP) as a cofactor in the active site. The MocR/GabR subfamily includes GabR and several other transcription factors that possess a Cterminal aminotransferase-like domain that binds PLP but is catalytically inactive and an N-terminal winged-helix domain that binds DNA [49][50][51][52][53][54][55][56]. It is likely that the DNA-binding domain was added to an ancestral aminotransferase enzyme to result in an intermediate moonlighting protein before loss of catalytic activity to result in the pseudoenzyme transcription factor. ...
As more genome sequences are elucidated, there is an increasing need for information about the functions of the millions of proteins they encode. The function of a newly sequenced protein is often estimated by sequence alignment with the sequences of proteins with known functions. However, protein superfamilies can contain members that share significant amino acid sequence and structural homology yet catalyze different reactions or act on different substrates. Some homologous proteins differ by having a second or even third function, called moonlighting proteins. More recently, it was found that most protein superfamilies also include pseudoenzymes, a protein, or a domain within a protein, that has a three‐dimensional fold that resembles a conventional catalytically active enzyme, but has no catalytic activity. In this review, we discuss several examples of protein families that contain enzymes, pseudoenzymes, and moonlighting proteins. It is becoming clear that pseudoenzymes and moonlighting proteins are widespread in the evolutionary tree, and in many protein families, and they are often very similar in sequence and structure to their monofunctional and catalytically active counterparts. A greater understanding is needed to clarify when similarities and differences in amino acid sequences and structures correspond to similarities and differences in biochemical functions and cellular roles. This information can help improve programs that identify protein functions from sequence or structure and assist in more accurate annotation of sequence and structural databases, as well as in our understanding of the broad diversity of protein functions.
