Loss-of-Function Mutations in APPL1 in Familial Diabetes Mellitus.

Diabetes mellitus is a highly heterogeneous disorder encompassing several distinct forms with different clinical manifestations including a wide spectrum of age at onset. Despite many advances, the causal genetic defect remains unknown for many subtypes of the disease, including some of those forms with an apparent Mendelian mode of inheritance. Here we report two loss-of-function mutations (c.1655T>A [p.Leu552(∗)] and c.280G>A [p.Asp94Asn]) in the gene for the Adaptor Protein, Phosphotyrosine Interaction, PH domain, and leucine zipper containing 1 (APPL1) that were identified by means of whole-exome sequencing in two large families with a high prevalence of diabetes not due to mutations in known genes involved in maturity onset diabetes of the young (MODY). APPL1 binds to AKT2, a key molecule in the insulin signaling pathway, thereby enhancing insulin-induced AKT2 activation and downstream signaling leading to insulin action and secretion. Both mutations cause APPL1 loss of function. The p.Leu552(∗) alteration totally abolishes APPL1 protein expression in HepG2 transfected cells and the p.Asp94Asn alteration causes significant reduction in the enhancement of the insulin-stimulated AKT2 and GSK3ß phosphorylation that is observed after wild-type APPL1 transfection. These findings-linking APPL1 mutations to familial forms of diabetes-reaffirm the critical role of APPL1 in glucose homeostasis.

Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor.

BACKGROUND: GabR is a transcriptional regulator belonging to the MocR/GabR family, characterized by a N-terminal wHTH DNA-binding domain and a C-terminal effector binding and/or oligomerization domain, structurally homologous to aminotransferases (ATs). In the presence of γ-aminobutyrate (GABA) and pyridoxal 5'-phosphate (PLP), GabR activates the transcription of gabT and gabD genes involved in GABA metabolism. METHODS: Here we report a biochemical and atomic force microscopy characterization of Bacillus subtilis GabR in complex with DNA. Complexes were assembled in vitro to study their stoichiometry, stability and conformation. RESULTS: The fractional occupancy of the GabR cognate site suggests that GabR binds as a dimer with Kd of 10nM. Upon binding GabR bends the DNA by 80° as measured by anomalous electrophoretic mobility. With GABA we observed a decrease in affinity and conformational rearrangements compatible with a less compact nucleo-protein complex but no changes of the DNA bending angle. By employing promoter and GabR mutants we found that basic residues of the positively charged groove on the surface of the AT domain affect DNA affinity. CONCLUSIONS: The present data extend current understanding of the GabR-DNA interaction and the effect of GABA and PLP. A model for the GabR-DNA complex, corroborated by a docking simulation, is proposed. GENERAL SIGNIFICANCE: Characterization of the GabR DNA binding mode highlights the key role of DNA bending and interactions with bases outside the canonical direct repeats, and might be of general relevance for the action mechanism of MocR transcription factors.

Hepatitis C virus genotype 3A in a population of injecting drug users in Montenegro: Bayesian and evolutionary analysis.

Few reports are available on HCV molecular epidemiology among IDUs in Eastern Europe, and none in Montenegro. The aim of this study was to investigate the HCV genotype distribution in Montenegro among IDUs and to perform Bayesian and evolutionary analysis of the most prevalent HCV genotype circulating in this population. Sixty-four HCV-positive IDUs in Montenegro were enrolled between 2013 and 2014, and the NS5B gene was sequenced. The Bayesian analysis showed that the most prevalent subtype was HCV-3a. Phylogenetic data showed that HCV-3a reached Montenegro in the late 1990s, causing an epidemic that exponentially grew between the 1995 and 2005. In the dated tree, four different entries, from 1990 (clade D), 1994 (clade A) to 1999 (clade B) and 2001 (clade C), were identified. In the NS5B protein model, the amino acids variations were located mainly in the palm domain, which contains most of the conserved structural elements of the active site. This study provides an analysis of the virus transmission pathway and the evolution of HCV genotype 3a among IDUs in Montenegro. These data could represent the basis for further strategies aimed to improve disease management and surveillance program development in high-risk populations.

On the mechanism of Escherichia coli pyridoxal kinase inhibition by pyridoxal and pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a crucial role in several cellular processes. In most organisms, PLP is recycled from nutrients and degraded B6-enzymes in a salvage pathway that involves pyridoxal kinase (PLK), pyridoxine phosphate oxidase and phosphatase activities. Regulation of the salvage pathway is poorly understood. Escherichia coli possesses two distinct pyridoxal kinases, PLK1, which is the focus of the present work, and PLK2. From previous studies dating back to thirty years ago, pyridoxal (PL) was shown to inhibit E. coli PLK1 forming a covalent link with the enzyme. This inhibition was proposed to play a regulative role in vitamin B6 metabolism, although its details had never been clarified. Recently, we have shown that also PLP produced during PLK1 catalytic cycle acts as an inhibitor, forming a Schiff base with Lys229, without being released in the solvent. The question arises as to which is the actual inhibition mechanism by PL and PLP. In the present work, we demonstrated that also PL binds to Lys229 as a Schiff base. However, the isolated covalent PLK1-PL complex is not inactive but, in the presence of ATP, is able to catalyse the single turnover production of PLP, which binds tightly to the enzyme and is ultimately responsible for its inactivation. The inactivation mechanism mediated by Lys229 may play a physiological role in controlling cellular levels of PLP. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Mutation of CHRNA2 in a family with benign familial infantile seizures: Potential role of nicotinic acetylcholine receptor in various phenotypes of epilepsy.

Nicotinic acetylcholine receptor genes are involved mainly in nocturnal frontal epilepsy. Despite extensive studies, to date, the α2 subunit did not show a strong association with this peculiar epileptic phenotype. We report CHRNA2 missense mutation in a family with benign familial infantile seizures (BFIS). TrueSeq Custom Amplicon (TSCA) sequencing approach was used to screen 10 ion channel genes in patients with idiopathic epilepsies. TSCA revealed a heterozygous single-nucleotide substitution in CHRNA2 gene (c.1126 C>T; p. Arg376Trp) that segregated in a family with BFIS; based on bio-informatics inspection, the change was predicted to be pathogenic. The investigated family includes parents and their three daughters. In affected individuals, seizures started between 6 and 24 months of age. Seizures were mainly in cluster and well-controlled. Outcome was good in all subjects. Even if nicotinic acetylcholine receptor genes are traditionally associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), this single-family description can open new possibilities in the genetic diagnosis, molecular characterization, and management of CHRNA2-related epilepsy. The pathogenic conversion of arginine 376 to tryptophan alters all of these interactions in the cytoplasmic domain, never reported to be involved in epileptogenic mechanism. Further functional tests will be necessary to strongly relate CHRNA2 mutation with BFIS phenotype.

Conformational transitions driven by pyridoxal-5'-phosphate uptake in the psychrophilic serine hydroxymethyltransferase from Psychromonas ingrahamii.

Serine hydroxymethyltransferase (SHMT) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme belonging to the fold type I superfamily, which catalyzes in vivo the reversible conversion of l-serine and tetrahydropteroylglutamate (H4PteGlu) to glycine and 5,10-methylenetetrahydropteroylglutamate (5,10-CH2-H4PteGlu). The SHMT from the psychrophilic bacterium Psychromonas ingrahamii (piSHMT) had been recently purified and characterized. This enzyme was shown to display catalytic and stability properties typical of psychrophilic enzymes, namely high catalytic activity at low temperature and thermolability. To gain deeper insights into the structure-function relationship of piSHMT, the three-dimensional structure of its apo form was determined by X-ray crystallography. Homology modeling techniques were applied to build a model of the piSHMT holo form. Comparison of the two forms unraveled the conformation modifications that take place when the apo enzyme binds its cofactor. Our results show that the apo form is in an "open" conformation and possesses four (or five, in chain A) disordered loops whose electron density is not visible by X-ray crystallography. These loops contain residues that interact with the PLP cofactor and three of them are localized in the major domain that, along with the small domain, constitutes the single subunit of the SHMT homodimer. Cofactor binding triggers a rearrangement of the small domain that moves toward the large domain and screens the PLP binding site at the solvent side. Comparison to the mesophilic apo SHMT from Salmonella typhimurium suggests that the backbone conformational changes are wider in psychrophilic SHMT.

Type I pyridoxal 5'-phosphate dependent enzymatic domains embedded within multimodular nonribosomal peptide synthetase and polyketide synthase assembly lines.

BACKGROUND: Pyridoxal 5'-phosphate (PLP)-dependent enzymes of fold type I, the most studied structural class of the PLP-dependent enzyme superfamily, are known to exist as stand-alone homodimers or homotetramers. These enzymes have been found also embedded in multimodular and multidomain assembly lines involved in the biosynthesis of polyketides (PKS) and nonribosomal peptides (NRPS). The aim of this work is to provide a proteome-wide view of the distribution and characteristics of type I domains covalently integrated in these assemblies in prokaryotes. RESULTS: An ad-hoc Hidden Markov profile was calculated using a sequence alignment derived from a multiple structural superposition of distantly related PLP-enzymes of fold type I. The profile was utilized to scan the sequence databank and to collect the proteins containing at least one type I domain linked to a component of an assembly line in bacterial genomes. The domains adjacent to a carrier protein were further investigated. Phylogenetic analysis suggested the presence of four PLP-dependent families: Aminotran_3, Beta_elim_lyase and Pyridoxal_deC, occurring mainly within mixed NRPS/PKS clusters, and Aminotran_1_2 found mainly in PKS clusters. Sequence similarity to the reference PLP enzymes with solved structures ranged from 24 to 42% identity. Homology models were built for each representative type I domain and molecular docking simulations with putative substrates were carried out. Prediction of the protein-protein interaction sites evidenced that the surface regions of the type I domains embedded within multienzyme assemblies were different from those of the self-standing enzymes; these structural features appear to be required for productive interactions with the adjacent domains in a multidomain context. CONCLUSIONS: This work provides a systematic view of the occurrence of type I domain within NRPS and PKS assembly lines and it predicts their structural characteristics using computational methods. Comparison with the corresponding stand-alone enzymes highlighted the common and different traits related to various aspects of their structure-function relationship. Therefore, the results of this work, on one hand contribute to the understanding of the functional and structural diversity of the PLP-dependent type I enzymes and, on the other, pave the way to further studies aimed at their applications in combinatorial biosynthesis.

Interaction of Bacillus subtilis GabR with the gabTD promoter: role of repeated sequences and effect of GABA in transcriptional activation.

Bacillus subtilis is able to use γ-aminobutyric acid (GABA) found in the soil as carbon and nitrogen source, through the action of GABA aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD). GABA acts as molecular effector in the transcriptional activation of the gabTD operon by GabR. GabR is the most studied member of the MocR family of prokaryotic pyridoxal 5'-phosphate (PLP)-dependent transcriptional regulators, yet crucial aspects of its mechanism of action are unknown. GabR binds to the gabTD promoter, but transcription is activated only when GABA is present. Here, we demonstrated, in contrast with what had been previously proposed, that three repeated nucleotide sequences in the promoter region, two direct repeats and one inverted repeat, are specifically recognized by GabR. We carried out in vitro and in vivo experiments using mutant forms of the gabTD promoter. Our results showed that GABA activates transcription by changing the modality of interaction between GabR and the recognized sequence repeats. A hypothetical model is proposed in which GabR exists in two alternative conformations that, respectively, prevent or promote transcription. According to this model, in the absence of GABA, GabR binds to DNA interacting with all three sequence repeats, overlapping the RNA polymerase binding site and therefore preventing transcription activation. On the other hand, when GABA binds to GabR, a conformational change of the protein leads to the release of the interaction with the inverted repeat, allowing transcription initiation by RNA polymerase.

A Comprehensive Computational Analysis of Mycobacterium Genomes Pinpoints the Genes Co-occurring with YczE, a Membrane Protein Coding Gene Under the Putative Control of a MocR, and Predicts its Function.

Bacterial proteins belonging to the YczE family are predicted to be membrane proteins of yet unknown function. In many bacterial species, the yczE gene coding for the YczE protein is divergently transcribed with respect to an adjacent transcriptional regulator of the MocR family. According to in silico predictions, proteins named YczR are supposed to regulate the expression of yczE genes. These regulators linked to the yczE genes are predicted to constitute a subfamily within the MocR family. To put forward hypotheses amenable to experimental testing about the possible function of the YczE proteins, a phylogenetic profile strategy was applied. This strategy consists in searching for those genes that, within a set of genomes, co-occur exclusively with a certain gene of interest. Co-occurrence can be suggestive of a functional link. A set of 30 mycobacterial complete proteomes were collected. Of these, only 16 contained YczE proteins. Interestingly, in all cases each yczE gene was divergently transcribed with respect to a yczR gene. Two orthology clustering procedures were applied to find proteins co-occurring exclusively with the YczE proteins. The reported results suggest that YczE may be involved in the membrane translocation and metabolism of sulfur-containing compounds mostly in rapidly growing, low pathogenicity mycobacterial species. These observations may hint at potential targets for therapies to treat the emerging opportunistic infections provoked by the widespread environmental mycobacterial species and may contribute to the delineation of the genomic and physiological differences between the pathogenic and non-pathogenic mycobacterial species.

Molecular dynamics simulation unveils the conformational flexibility of the interdomain linker in the bacterial transcriptional regulator GabR from Bacillus subtilis bound to pyridoxal 5'-phosphate.

GabR from Bacillus subtilis is a transcriptional regulator belonging to the MocR subfamily of the GntR regulators. The structure of the MocR regulators is characterized by the presence of two domains: i) a N-terminal domain, about 60 residue long, possessing the winged-Helix-Turn-Helix (wHTH) architecture with DNA recognition and binding capability; ii) a C-terminal domain (about 350 residue) folded as the pyridoxal 5'-phosphate (PLP) dependent aspartate aminotransferase (AAT) with dimerization and effector binding functions. The two domains are linked to each other by a peptide bridge. Although structural and functional characterization of MocRs is proceeding at a fast pace, virtually nothing is know about the molecular changes induced by the effector binding and on how these modifications influence the properties of the regulator. An extensive molecular dynamics simulation on the crystallographic structure of the homodimeric B. subtilis GabR has been undertaken with the aim to envisage the role and the importance of conformational flexibility in the action of GabR. Molecular dynamics has been calculated for the apo (without PLP) and holo (with PLP bound) forms of the GabR. A comparison between the molecular dynamics trajectories calculated for the two GabR forms suggested that one of the wHTH domain detaches from the AAT-like domain in the GabR PLP-bound form. The most evident conformational change in the holo PLP-bound form is represented by the rotation and the subsequent detachment from the subunit surface of one of the wHTH domains. The movement is mediated by a rearrangement of the linker connecting the AAT domain possibly triggered by the presence of the negative charge of the PLP cofactor. This is the second most significant conformational modification. The C-terminal section of the linker docks into the "active site" pocket and establish stabilizing contacts consisting of hydrogen-bonds, salt-bridges and hydrophobic interactions.

Salmonella typhimurium PtsJ is a novel MocR-like transcriptional repressor involved in regulating the vitamin B6 salvage pathway.

The vitamin B6 salvage pathway, involving pyridoxine 5'-phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B6 vitamers from nutrients and protein turnover to produce pyridoxal 5'-phosphate (PLP), the catalytically active form of the vitamin. Regulation of this pathway, widespread in living organisms including humans and many bacteria, is very important to vitamin B6 homeostasis but poorly understood. Although some information is available on the enzymatic regulation of PNPOx and PLK, little is known on their regulation at the transcriptional level. In the present work, we identified a new MocR-like regulator, PtsJ from Salmonella typhimurium, which controls the expression of the pdxK gene encoding one of the two PLKs expressed in this organism (PLK1). Analysis of pdxK expression in a ptsJ knockout strain demonstrated that PtsJ acts as a transcriptional repressor. This is the first case of a MocR-like regulator acting as repressor of its target gene. Expression and purification of PtsJ allowed a detailed characterisation of its effector and DNA-binding properties. PLP is the only B6 vitamer acting as effector molecule for PtsJ. A DNA-binding region composed of four repeated nucleotide sequences is responsible for binding of PtsJ to its target promoter. Analysis of binding stoichiometry revealed that protein subunits/DNA molar ratio varies from 4 : 1 to 2 : 1, depending on the presence or absence of PLP. Structural characteristics of DNA transcriptional factor-binding sites suggest that PtsJ binds DNA according to a different model with respect to other characterised members of the MocR subgroup.

A Bioinformatics Analysis Reveals a Group of MocR Bacterial Transcriptional Regulators Linked to a Family of Genes Coding for Membrane Proteins.

The MocR bacterial transcriptional regulators are characterized by an N-terminal domain, 60 residues long on average, possessing the winged-helix-turn-helix (wHTH) architecture responsible for DNA recognition and binding, linked to a large C-terminal domain (350 residues on average) that is homologous to fold type-I pyridoxal 5'-phosphate (PLP) dependent enzymes like aspartate aminotransferase (AAT). These regulators are involved in the expression of genes taking part in several metabolic pathways directly or indirectly connected to PLP chemistry, many of which are still uncharacterized. A bioinformatics analysis is here reported that studied the features of a distinct group of MocR regulators predicted to be functionally linked to a family of homologous genes coding for integral membrane proteins of unknown function. This group occurs mainly in the Actinobacteria and Gammaproteobacteria phyla. An analysis of the multiple sequence alignments of their wHTH and AAT domains suggested the presence of specificity-determining positions (SDPs). Mapping of SDPs onto a homology model of the AAT domain hinted at possible structural/functional roles in effector recognition. Likewise, SDPs in wHTH domain suggested the basis of specificity of Transcription Factor Binding Site recognition. The results reported represent a framework for rational design of experiments and for bioinformatics analysis of other MocR subgroups.

Data from computational analysis of the peptide linkers in the MocR bacterial transcriptional regulators.

Detailed data from statistical analyses of the structural properties of the inter-domain linker peptides of the bacterial regulators of the family MocR are herein reported. MocR regulators are a recently discovered subfamily of bacterial regulators possessing an N-terminal domain, 60 residue long on average, folded as the winged-helix-turn-helix architecture responsible for DNA recognition and binding, and a large C-terminal domain (350 residue on average) that belongs to the fold type-I pyridoxal 5'-phosphate (PLP) dependent enzymes such aspartate aminotransferase. Data show the distribution of several structural characteristics of the linkers taken from bacterial species from five different phyla, namely Actinobacteria, Alpha-, Beta-, Gammaproteobacteria and Firmicutes. Interpretation and discussion of reported data refer to the article "Structural properties of the linkers connecting the N- and C- terminal domains in the MocR bacterial transcriptional regulators" (T. Milano, S. Angelaccio, A. Tramonti, M. L. Di Salvo, R. Contestabile, S. Pascarella, 2016) [1].

Structural properties of the linkers connecting the N- and C- terminal domains in the MocR bacterial transcriptional regulators.

Peptide inter-domain linkers are peptide segments covalently linking two adjacent domains within a protein. Linkers play a variety of structural and functional roles in naturally occurring proteins. In this work we analyze the sequence properties of the predicted linker regions of the bacterial transcriptional regulators belonging to the recently discovered MocR subfamily of the GntR regulators. Analyses were carried out on the MocR sequences taken from the phyla Actinobacteria, Firmicutes, Alpha-, Beta- and Gammaproteobacteria. The results suggest that MocR linkers display phylum-specific characteristics and unique features different from those already described for other classes of inter-domain linkers. They show an average length significantly higher: 31.8 ± 14.3 residues reaching a maximum of about 150 residues. Compositional propensities displayed general and phylum-specific trends. Pro is dominating in all linkers. Dyad propensity analysis indicate Pro-Pro as the most frequent amino acid pair in all linkers. Physicochemical properties of the linker regions were assessed using amino acid indices relative to different features: in general, MocR linkers are flexible, hydrophilic and display propensity for ß-turn or coil conformations. Linker sequences are hypervariable: only similarities between MocR linkers from organisms related at the level of species or genus could be found with sequence searches. The results shed light on the properties of the linker regions of the new MocR subfamily of bacterial regulators and may provide knowledge-based rules for designing artificial linkers with desired properties.

Zika Virus spreading in South America: Evolutionary analysis of emerging neutralizing resistant Phe279Ser strains.

OBJECTIVE: To investigate the genetic diversity of Zika Virus (ZIKV) and the relationships existing among these circulating viruses worldwide. To evaluate the genetic polymorphisms harbored from ZIKV that can have an influence on the virus circulation. METHODS: Three different ZIKV dataset were built. The first dataset included 63 E gene sequences, the second one 22 NS3 sequences and the third dataset was composed of 108 NS5 gene sequences. Phylogenetic and selective pressure analysis was performed. The edited nucleic acid alignment from the Envelope dataset was used to generate a conceptual translation to the corresponding peptide sequences through UGene software. RESULTS: The phylogeographic reconstruction was able to discriminate unambiguously that the Brazilian strains are belonged to the Asian lineage. The structural analysis reveals instead the presence of the Ser residue in the Brazilian sequences (however already observed in other previously reported ZIKV infections) that could suggest the presence of a neutralization-resistant population of viruses. CONCLUSIONS: Phylogenetic, evolutionary and selective pressure analysis contributed to improve the knowledge on the circulation of ZIKV.

Klebsiella pneumoniae blaKPC-3 nosocomial epidemic: Bayesian and evolutionary analysis.

K. pneumoniae isolates carrying blaKPC-3 gene were collected to perform Bayesian phylogenetic and selective pressure analysis and to apply homology modeling to the KPC-3 protein. A dataset of 44 blakpc-3 gene sequences from clinical isolates of K. pneumoniae was used for Bayesian phylogenetic, selective pressure analysis and homology modeling. The mean evolutionary rate for blakpc-3 gene was 2.67×10-3 substitution/site/year (95% HPD: 3.4×10-4-5.59×10-3). The root of the Bayesian tree dated back to the year 2011 (95% HPD: 2007-2012). Two main clades (I and II) were identified. The population dynamics analysis showed an exponential growth from 2011 to 2013 and the reaching of a plateau. The phylogeographic reconstruction showed that the root of the tree had a probable common ancestor in the general surgery ward. Selective pressure analysis revealed twelve positively selected sites. Structural analysis of KPC-3 protein predicted that the amino acid mutations are destabilizing for the protein and could alter the substrate specificity. Phylogenetic analysis and homology modeling of blaKPC-3 gene could represent a useful tool to follow KPC spread in nosocomial setting and to evidence amino acid substitutions altering the substrate specificity.

Conserved water molecules in bacterial serine hydroxymethyltransferases.

Water molecules occurring in the interior of protein structures often are endowed with key structural and functional roles. We report the results of a systematic analysis of conserved water molecules in bacterial serine hydroxymethyltransferases (SHMTs). SHMTs are an important group of pyridoxal-5'-phosphate-dependent enzymes that catalyze the reversible conversion of l-serine and tetrahydropteroylglutamate to glycine and 5,10-methylenetetrahydropteroylglutamate. The approach utilized in this study relies on two programs, ProACT2 and WatCH. The first software is able to categorize water molecules in a protein crystallographic structure as buried, positioned in clefts or at the surface. The other program finds, in a set of superposed homologous proteins, water molecules that occur approximately in equivalent position in each of the considered structures. These groups of molecules are referred to as 'clusters' and represent structurally conserved water molecules. Several conserved clusters of buried or cleft water molecules were found in the set of 11 bacterial SHMTs we took into account for this work. The majority of these clusters were not described previously. Possible structural and functional roles for the conserved water molecules are envisaged. This work provides a map of the conserved water molecules helpful for deciphering SHMT mechanism and for rational design of molecular engineering experiments.

The aspartate aminotransferase-like domain of Firmicutes MocR transcriptional regulators.

Bacterial MocR transcriptional regulators possess an N-terminal DNA-binding domain containing a conserved helix-turn-helix module and an effector-binding and/or oligomerization domain at the C-terminus, homologous to fold type-I pyridoxal 5'-phosphate (PLP) enzymes. Since a comprehensive structural analysis of the MocR regulators is still missing, a comparisons of Firmicutes MocR sequences was undertook to contribute to the understanding of the structural characteristics of the C-terminal domain of these proteins, and to shed light on the structural and functional relationship with fold type-I PLP enzymes. Results of this work suggest the presence of at least three subgroups within the MocR sequences and provide a guide for rational site-directed mutagenesis studies aimed at deciphering the structure-function relationships in this new protein family.

Molecular mechanism of PdxR ­ a transcriptional activator involved in the regulation of vitamin B6 biosynthesis in the probiotic bacterium Bacillus clausii.

Pyridoxal 5'-phosphate (PLP), the well-known active form of vitamin B6 , is an essential enzyme cofactor involved in a large number of metabolic processes. PLP levels need to be finely tuned in response to cell requirements; however, little is known about the regulation of PLP biosynthesis and recycling pathways. The transcriptional regulator PdxR activates transcription of the pdxST genes encoding PLP synthase. It is characterized by an N-terminal helix-turn-helix motif that binds DNA and an effector-binding C-terminal domain homologous to PLP-dependent enzymes. Although it is known that PLP acts as an anti-activator, the mechanism of action of PdxR is unknown. In the present study, we analyzed the biochemical and DNA-binding properties of PdxR from the probiotic Bacillus clausii. Spectroscopic measurements showed that PLP is the only B6 vitamer that acts as an effector molecule of PdxR. Binding of PLP to PdxR determines a protein conformational change, as detected by gel filtration chromatography and limited proteolysis experiments. We showed that two direct repeats and one inverted repeat are present in the DNA promoter region and PdxR is able to bind DNA fragments containing any combination of two of them. However, when PLP binds to PdxR, it modifies the DNA-binding properties of the protein, making it selective for inverted repeats. A molecular mechanism is proposed in which the two different DNA binding modalities of PdxR determined by the presence or absence of PLP are responsible for the control of pdxST transcription.