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A Survey of Pyridoxal 5′-Phosphate-Dependent Proteins in the Gram-Positive Model Bacterium Bacillus subtilis

The B6 vitamer pyridoxal 5′-phosphate (PLP) is a co-factor for proteins and enzymes that are involved in diverse cellular processes. Therefore, PLP is essential for organisms from all kingdoms of life. Here we provide an overview about the PLP-dependent proteins from the Gram-positive soil bacterium Bacillus subtilis. Since B. subtilis serves as a model system in basic research and as a production host in industry, knowledge about the PLP-dependent proteins could facilitate engineering the bacteria for biotechnological applications. The survey revealed that the majority of the PLP-dependent proteins are involved in metabolic pathways like amino acid biosynthesis and degradation, biosynthesis of antibacterial compounds, utilization of nucleotides as well as in iron and carbon metabolism. Many PLP-dependent proteins participate in de novo synthesis of the co-factors biotin, folate, heme, and NAD+ as well as in cell wall metabolism, tRNA modification, regulation of gene expression, sporulation, and biofilm formation. A surprisingly large group of PLP-dependent proteins (29%) belong to the group of poorly characterized proteins. This review underpins the need to characterize the PLP-dependent proteins of unknown function to fully understand the “PLP-ome” of B. subtilis.

The DXP-independent vitamin B6 biosynthetic pathway involves only the PdxST enzyme complex (Ehrenshaft and Daub, 2001;Belitsky, 2004b;Burns et al., 2005;Raschle et al., 2005;Strohmeier et al., 2006). PdxT is a glutaminase that hydrolyses glutamine into glutamate and ammonium, of which the latter serves as a substrate to the PLP synthase PdxS (Belitsky, 2004b). The PdxS subunit generates PLP from ammonium together with either ribulose 5-phosphate or ribose 5-phosphate and with either G3P or dihydroxyacetone phosphate ( Figure 1B). Many organisms possess a salvage pathway for the interconversion of the B6 vitamers ( Figure 1B) (Fitzpatrick et al., 2007;di Salvo et al., 2011). For instance, specific B6-vitamer kinases can phosphorylate PN, PM and PL into their respective phosphate esters (White and Dempsey, 1970;Yang et al., 1996Yang et al., , 1998di Salvo et al., 2004;Park et al., 2004). Those organisms carrying only salvage pathways have to take up B6 vitamers. So far, only few vitamin B6 transporters have been in described in eukaryotes (Stolz and Vielreicher, 2003;Szydlowski et al., 2013). The accumulation of PLP to toxic levels can be prevented by dephosphorylation and export of PL. Indeed, bacteria like E. coli and Sinorhizobium meliloti synthesize a phosphatase for dephosphorylation of PNP and PLP (Tazoe et al., 2005;Nagahashi et al., 2008;Sugimoto et al., 2017).
In contrast to the enzymes of the DXP-dependent vitamin B6 pathway from E. coli, the PdxST enzyme complex from B. subtilis is rather slow (Rosenberg et al., 2017). Therefore, a heterologous DXP-dependent vitamin B6 pathway has been introduced into B. subtilis for producing the B6 vitamer PN (Commichau et al., 2014(Commichau et al., , 2015. The fact that the engineered B. subtilis strains synthesized significant amounts of PN, which was detectable in the culture supernatant, suggests that the bacteria might possess a PNP phosphatase and an export system for PN. Recently, the PLP phosphatase YbhA has been identified in E. coli (Sugimoto et al., 2017). YbhA shows about 31% overall sequence identity with the YitU protein from B. subtilis. Even though it has been shown that YitU is a HAD phosphatase having a minor activity in dephosphorylating the riboflavin precursor 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5 ′ -phosphate (Sarge et al., 2015), it will be interesting to test whether the protein may act as a PNP/PLP phosphatase.

TOXICITY OF VITAMIN B6 AND PATHWAY INTERMEDIATES
PLP can be toxic for the cell because the reactive 4 ′ -aldehyde moiety of the B6 vitamer forms covalent adducts with other compounds and PLP-independent proteins containing thiol or amino groups. For instance, PLP was shown to inhibit enzymes that are involved in DNA metabolism and in central carbon metabolism in eukaryotes (Mizushina et al., 2003;Vermeersch et al., 2004;Lee et al., 2005). Moreover, the modification of the E. coli initiation factor 3, the adenylsuccinate synthetase and the PL kinase by PLP results in activity loss (Ohsawa and Gualerzi, 1981;Dong and Fromm, 1990;Ghatge et al., 2012). Recently, it has been shown that the addition of vitamin B6 to the E. coli wild type strain BW25113 and an E. coli mutant strain lacking the ZipA cell division protein affects multiple metabolic pathways, which are involved in amino acid biosynthesis (Vega and Margolin, 2017). Thus, excess of PLP affects different cellular processes. PLP is also prone to damage either due to side reactions that are catalyzed by promiscuous enzymes or due to spontaneous chemical reactions (Linster et al., 2013). In fact, the B6 vitamers PLP and PMP were identified as members of the 30 most damage-prone metabolites (Lerma-Ortiz et al., 2016). However, given the fact that PLP is required for optimal growth in little amounts, the essential cofactor can be synthesized at a minimal necessary rate (Hartl et al., 2017). The low requirement of PLP and its low cellular concentration prevent perturbation of other essential processes in the cell.
In the past years, several attempts have been made to engineer bacteria for the overproduction of the B6 vitamers PL and PN (Rosenberg et al., 2017). In contrast to PL(P), PN(P) is less toxic for living cells (see above; Commichau et al., 2014). Therefore, the DXP-dependent seems to be a promising pathway for engineering bacteria for vitamin B6 overproduction. However, it has also been shown that intermediates of the DXP-dependent pathway can be highly toxic for bacteria. For instance, the erythrose-4-phosphate dehydrogenase Epd generates the DXP-dependent vitamin B6 pathway intermediate 4-phosphoerythronate (4PE), which is required in low amounts for PLP biosynthesis, is toxic for the cells when overproduced (Sachla and Helmann, 2019). Eukaryotic cells do have a phosphatase that hydrolyzes and detoxifies 4PE that is also mistakenly generated by the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Beaudoin and Hanson, 2016;Collard et al., 2016). 4PE inihibits the 6-phosphogluconate dehydrogenase from the pentosephosphate pathway (Collard et al., 2016). Recently, it has been demonstrated that 4PE also inhibits the B. subtilis 6-phosphogluconate dehydrogenase GndA (Sachla and Helmann, 2019). In this organism, 4PE is detoxified by the GTPase CpgA, which is a checkpoint protein known to be involved in ribosome assembly (Campbell et al., 2005). It will be interesting to assess whether 4PE also inhibits the 6-phosphogluconate dehydrogenase in E. coli because the bacterium does not possess a CpgA homolog. However, the accumulation of 4PE to toxic levels does not seem to be problematic in E. coli because 4PE can be produced in only small amounts that are sufficient for de novo synthesis of PLP. The intermediate 4HTP from the DXP-dependent vitamin B6 pathway is also inhibits bacterial growth. 4HTP interferes with biosynthesis of threonine and isoleucine in E. coli and B. subtilis (Drewke et al., 1993;Farrington et al., 1993;Commichau et al., 2014Commichau et al., , 2015Rosenberg et al., 2016). The toxicity of the pathway intermediates may explain why it is difficult to engineer bacteria that stably express the genes of the DXP-dependent vitamin B6 pathway (Commichau et al., 2014). The understanding of the metabolite toxicity is crucial for the rational design and engineering of bacteria overproducing PN at commercially attractive levels. Moreover, the knowledge about the functions of target proteins of PLP is very important to understand how the B6 vitamer affects cellular metabolism upon overproduction.

PLP-DEPENDENT PROTEINS AND ENZYMES INVOLVED IN VITAMIN B6 METABOLISM IN BACILLUS SUBTILIS
To identify the B. subtilis proteins and enzymes that require PLP for activity and are involved in vitamin B6 metabolism, we compared the Enzyme Commission (E.C.) numbers of the proteins from the B. subtilis 168 laboratory strain found in the SubtiWiki database (http://subtiwiki.uni-goettingen.de/v3/) (Zhu and Stülke, 2017) with the E.C. numbers that are deposited in the B6 database (Table 1) (http://bioinformatics.unipr.it/cgi-bin/ bioinformatics/B6db/home.pl) (Percudani and Peracchi, 2009). We also describe proteins from the SubtiWiki database that are specific for B. subtilis and are therefore not present in the B6 database. Publications describing proteins involved in vitamin B6 metabolism in B. subtilis were also added to the Table. A recent mass spectrometry approach in combination with modified pyridoxal analogs identified proteins in the Grampositive pathogen Staphylococcus aureus that probably depend on the B6 vitamer PLP (Hoegl et al., 2018). The study confirmed the binding of PLP to proteins of known and unknown function and identified 4 additional PLP-binding proteins (HemH, HemQ, YtoP, and YwlG) (see below). In total we ended up with 65 PLP-dependent proteins in B. subtilis, of which 61 proteins are bona fide PLP-dependent proteins. The PLP-dependency of four proteins remains to be experimentally validated. Table 1 also contains the PDB identifiers of structures that are available in the PDB database for the B. subtilis proteins. In case the structural information was not available, we have added the PDB identifiers from PLP-dependent homologs showing more than 27% overall sequence identity. We have also included information about the physiological functions of the proteins and their paralogs, the transcription factors that are involved in synthesis of the proteins and information about the sequence similarities with other proteins from the UniProt database (https://www.uniprot. org). The list of proteins involved in vitamin B6 metabolism in B. subtilis will certainly be extended in the future because PLPdependent enzymes are ubiquitous and evolutionary diverse, making their classification based on sequence homology difficult.

FUNCTIONAL ASSIGNMENT OF KNOWN PLP-DEPENDENT PROTEINS IN B. SUBTILIS
Most of the proteins that require PLP in B. subtilis are metabolic enzymes, of which the majority is involved in anabolism and catabolism of proteinogenic and non-proteinogenic amino acids (Table 1; Figure 2). The enzymes can be assigned to known protein families of PLP-dependent enzymes and for most of them it has been shown that they are indeed active in amino acid metabolism (Mehta et al., 1993;Mehta and Christen, 2000) (Table 1). B. subtilis also possesses a PLPdependent 2-amino-3-ketobutyrate CoA ligase (Kbl), which could be involved in threonine utilization together with the Lthreonine dehydrogenase Tdh (Schmidt et al., 2001;Reitzer, 2005). Both enzymes are encoded in the bicistronic tdh-kbl operon (Nicolas et al., 2012). The regulation of the tdh and kbl genes and the catalytic activities of the Tdh and Kbl enzymes remain to be studied. Two PLP-dependent enzymes BacF and NtdA are involved in the synthesis of bacilysin and kanosamine in B. subtilis. Bacilysin is a non-ribosomally synthesized peptide that is active against various bacteria and some fungi (Inaoka et al., 2003(Inaoka et al., , 2009Karatas et al., 2003;Köroglu et al., 2011). Kanosamine is an antibiotic, which is produced by Bacillus A large-scale mass spectrometry-based screen revealed that the proteins probably bind PLP in S. aureus (Hoegl et al., 2018). It has to be experimentally validated that HemH, HemQ, YtoP and YwlG are functional PLP-dependent proteins.
Frontiers in Molecular Biosciences | www.frontiersin.org  Table 1 for more information.
and Streptomyces species and inhibits cell wall synthesis in microorganisms (Dolak et al., 1980;Milner et al., 1996;Inaoka et al., 2004;Inaoka and Ochi, 2011;Vetter et al., 2013). The PLP-dependent enzymes SufS, GlgP, and PucG are involved in iron-sulfur cluster formation, glycogen biosynthesis, and purine utilization, respectively ( Table 1). Homologs of SufS and GlgP are also present in E. coli (48 and 44% overall sequence identity, respectively). However, in B. subtilis the glycogen phosphorylase seems to be involved in a sporulation-specific process because the glgP gene is expressed early during sporulation in the mother cell (Kiel et al., 1994). Glycogen biosynthesis exclusively occurs in the presence of carbon sources allowing efficient sporulation (Kiel et al., 1994). Eight PLP-dependent enzymes are involved in the biosynthesis of the co-factors biotin, folate, heme and NAD (Table 1). While the biochemical and structural characterization of BioA, BioF, PabC, GsaB, HemL, and NifS revealed that the proteins require PLP for enzyme activity, it has to be investigated whether HemH and HemQ are bona fide PLP-dependent proteins. HemH and HemQ were recently identified in a mass spectrometry approach in the Gram-positive pathogen Staphylococcus aureus (Hoegl et al., 2018). B. subtilis possesses in total five PLP-dependent enzymes (Alr, Dat, PatA, NifZ, and YrvO) that are involved in cell wall metabolism and in information processing ( Table 1). The alanine racemase Alr, the D-alanine aminotransferase Dat and the N-acetyl-L,Ldiaminopimelate aminotransferase PatA generate precursors for the peptidoglycan of the cell wall. The tRNA-modifing enzymes NifZ and YrvO are both cysteine desulfurases that are active in biosynthesis of 4-thiouridine and 2-thiouridine, respectively, for the formation of modified tRNA molecules. YrvO transfers sulfur to the TrmU tRNA methyltransferase, which is essential for 2thiouridine biosynthesis (Black and Dos Santos, 2015). Finally, four PLP-dependent enzymes play a role in sporulation and biofilm formation in B. subtilis (Table 1). While it has been shown that Sps proteins such as SpsC are required for spore germination (Cangiano et al., 2014), the role of the alanine racemase YncD in sporulation is currently unkown. The two biofilm-related enzymes, the arginine decarboxylases SpeA and the UDP-2,6dideoxy-2-acetamido-4-keto-glucose aminotransferase EpsN are important for biosynthesis of polyamines such as spermidine and extracellular polysaccharides (Burrell et al., 2010;Marvasi et al., 2010). Indeed, B. subtilis strains lacking either SpeA or EpsN are defective in biofilm formation (Burrell et al., 2010;Pozsgai et al., 2012). SpeA also possesses a paralog (YaaO, 34% overall sequence identity), However, this proteins does not seem to be involved in biofilm formation (see below). To conclude, B. subtilis possesses several PLP-dependent enzymes that are involved in different cellular processes. Moreover, many PLP-dependent enzymes do have paralogs that have similar activities or fulfill specific functions in the cell, probably due to specialization during evolution (Table 1).

PLP-DEPENDENT TRANSCRIPTION FACTORS IN BACILLUS SUBTILIS
B. subtilis possesses seven PLP-dependent DNA-binding transcription factors of which only one has been intensively characterized (see below; Table 1). The PLP-dependent transcription factors belong to the MocR-subfamily and contain a GntR-family DNA-binding domain at the N-terminus and an aminotransferase-like sensory domain at the C-terminus (Bramucci et al., 2011;Milano et al., 2015;Tramonti et al., 2015Tramonti et al., , 2017Suvorova and Rodionov, 2016). The MocR-familytype PLP-dependent transcription factors that have been characterized are involved in controlling the expression of genes involved in PLP, γ-aminobutyrate, ecotoine, and taurine metabolism (Suvorova and Rodionov, 2016;Schulz et al., 2017;Tramonti et al., 2018). B. subtilis can utilize γ-aminobutyrate (GABA) as a source of nitrogen (Belitsky and Sonenshein, 2002). The catabolism of GABA requires the activities of the GABA aminotransferase GabT and the succinic semi-aldehyde dehydrogenase GabD that are encoded in the bicistronic gabT-gabD operon. The MocR-family-type regulator GabR activates the transcription of the gabT and gabD genes in a PLPand GABA-dependent manner (Belitsky and Sonenshein, 2002;Belitsky, 2004a). The protein also binds to the gabR promoter and represses gabR transcription in the absence of GABA (Belitsky and Sonenshein, 2002;Edayathumangalam et al., 2013). Structual analysis of GabR revealed that the aminotransferase-like activity at the C-terminus of the protein is not essential for its function as a transcription regulator (Edayathumangalam et al., 2013). It has also been shown that the formation of an external aldimine between GABA and PLP causes a conformational transition from the open form to a closed form in the aminotransferase domain of GabR, triggering transcription activation of the gabT-gabD operon (Okuda et al., 2015a;Milano et al., 2017;Park et al., 2017;Wu et al., 2017). Moreover, the dimerization of the C-terminal domain of GabR enables the N-terminal domain to bind to DNA and the N-terminal domain controls the binding specificity of the effector domain (Okuda et al., 2015b). The analysis of the interaction between GabR and DNA revealed that two cognate binding sites and the bendability of the interjacent DNA sequence are important for transcription factor binding (Al-Zyoud et al., 2016;Amidani et al., 2017). To conclude, the biochemical and structural characterization of the B. subtilis GabR transcriptional regulator has uncovered mechanistic insights into the MocRfamily-type PLP-dependent transcription factors and provides the basis for the characterization of the remaining six putative PLP-dependent DNA-binding transcription factors YcxD, YdeF, YdeL, YdfD, YhdI, and YisV from B. subtilis. Their characterization will presumably uncover novel metabolic pathways and transport systems (Suvorova and Rodionov, 2016).

POORLY CHARACTERIZED PLP-DEPENDENT ENZYMES
In addition to the 6 transcription factors whose DNA-binding activities depend on PLP and additional unknown effectors (see above), B. subtilis possesses several poorly characterized PLPdependent enzymes (Table 1; Figure 2). The DsdA protein from B. subtilis shows about 58% overalls sequence identity with the E. coli D-serine deaminase (D-serine ammonia lyase) DsdA, which catalyzes the deamination of D-serine to form pyruvate and ammonia (Gale and Stephenson, 1938;McFall, 1964). The PLP-dependency and the structure of the enzyme from E. coli have been determined (Schnackerz et al., 1999;Urusova et al., 2012). Phylogenetic analyses suggest that the E. coli and B. subtilis D-serine deaminases and threonine synthases with similarities in the catalytic mechanisms may have evolved from a common ancestor (Parsot, 1986). The primary function of DsdA seems to be the detoxification of D-serine, which inhibits bacterial growth because it is a competitive antagonist of β-alanine in the pantothenate (vitamin B5) biosynthetic pathway, generating the precursor for coenzyme A biosynthesis (Cosloy and McFall, 1973). Previously, it has also been shown that E. coli mutants constitutively expressing DsdA are able to use D-serine as the sole source of carbon and nitrogen (Bloom and McFall, 1975). In B. subtlilis the dsdA gene is located in the yqjP-yqjQ-dsdA-coaA-yqjT operon, containing three genes of unknown function as well as the dsdA and coaA genes, of which the latter encodes the major pantothenate kinase CoaA (Ogata et al., 2014). The genetic context of the dsdA gene strongly suggests that the D-serine deaminase may be involved in the detoxification of D-serine that probably also interferes with pantothenate synthesis in B. subtilis. The presence of a D-serine deaminase may be explained by the fact that L-serine is more rapidly racemized than most other amino acids (Reitzer, 2005). It will be interesting to elucidate whether overproduction of DsdA allows B. subtilis to grow with D-serine as the sole source of carbon and nitrogen and whether DsdA is involved in the detoxification of D-serine. The B. subtilis KamA enzyme shows about 60% overalls sequence identity with the PLP-, S-adenosyl-L-methionine and [4Fe-4S]-dependent lysine-2,3-aminomutase from the obligate anaerobe bacterium Clostridium subterminale ( Table 1) (Lepore et al., 2005). The lysine-2,3-aminomutase catalyzes the converstion of L-lysine to L-β-lysine, which is the first step in the anaerobic degradation of lysine in clostridia (Chirpich et al., 1970). In vitro characterization of KamA from B. subtilis revealed that enzyme also catalyzes the conversion of L-lysine to L-βlysine under anaerobic conditions (Chen et al., 2000). The KamA enzyme is only produced during sporulation of B. subtilis (Feucht et al., 2003). Therefore, the enzyme does not seem to play a role during vegetative growth. The lysine-2,3-aminomutase EpmB from E. coli, which shows about 31% overalls sequence identity with KamA from B. subtilis, has low lysine-2,3-aminomutase activity, indicating that L-lysine does not seem to be the natural substrate (Chen et al., 2000;Yanagisawa et al., 2010). Recently, it has been shown that the E. coli lysine-2,3-aminomutase EpmB enhances the lysylation of the elongation factor EF-P by the aminoacyl-tRNA synthetase GenX (Yanagisawa et al., 2010). The lysylation of EF-P is a post-translational modification that is essential for cell survival (Yanagisawa et al., 2010;Park et al., 2012). However, the physiological function of the lysine-2,3-aminomutase KamA from B. subtilis remains to be determined.
The B. subtilis YaaO enzyme, which belongs to class I Orn/Lys/Arg decarboxylases, encodes a putative arginine decarboxylase ( Table 1). Arginine decarboxylases are important for biosynthesis of polyamines such as spermidine, substances that are crucial for biofilm formation (Burrell et al., 2010). The B. subtilis arginine decarboxylase SpeA, which can be considered as a paralog of YaaO (34% overall sequence identity), is indeed essential for the synthesis of polyamines and thus biofilm formation (see above) (Burrell et al., 2010). However, no biofilmrelated phenotype has been reported so far for a B. subtilis mutant lacking YaaO. Recently, it has been reported that YaaO and two other proteins of unknown function (YfkA and YwlG, see below) influence the level of the post-translational aminopentanol modification of the elongation factor EF-P (Witzky et al., 2018). However, the precise role of YaaO in the modification of EF-P in B. subtilis remains to be elucidated.
The uncharacterized PLP-dependent proteins YcbU, AlaT, YhxA, and its paralog YodT are probably PLP-dependent amino acid transferases (Table 1). YcbU might be a cysteine desulfurase that is involved in co-factor biosynthesis (Mihara and Esaki, 2002). However, it remains to be elucidated whether YcbU is functional in B. subtilis because a mutant lacking YcbU shows no obvious phenotype (Koo et al., 2017). AlaT is similar to PLP-dependent methionine amino acid transferases and the protein shares about 47% overall sequence identity with an amino acid transferase from Pyrococcus horikoshii that acts on aromatic amino acids (Matsui et al., 2000). However, not experimental evidence supporting the annotation of AlaT is available. Both, YhxA and YodT are annotated as putative adenosylmethionine-8-amino-7-oxononanoate aminotransferases, enzymes that were shown to be involved in biotin biosynthesis in bacteria (Izumi et al., 1975). YhxA shares about 35 and 33% sequence identity with YodT and BioA, respectively. The PLP-dependent lysine-8-amino-7-oxononanoate aminotransferase BioA is required for biotin biosynthesis in B. subtilis (Table 1). Therefore, it is tempting to speculate that YhxA and YodT are also involved in biotin metabolism in this organism. The expression of the yhxA and yodT genes depends on SigA and on the sporulationspecific sigma factor SigE, respectively. Therefore, these enzymes seem to be active in different cellular differentiation processes of B. subtilis.
The B. subtilis YlmE protein of unknown function shows about 33% overall sequence identity with the YggS protein from E. coli. Recently, it has been shown that YggS is a PLP-binding protein, which belongs to a highly conserved COG0325 protein family and exists in almost all kingdoms of life, including bacteria, fungi and animals (Ito et al., 2013). The high conservation of YggS indicates that the protein fulfills an important function in bacteria. Indeed, the lack of YggS in E. coli affects balance of PLP homeostasis, sensitivity toward the B6 vitamer PN and perturbation of biosynthesis of branched-chain amino acids . Similar phenotypes have been associated to a mutant strain of Synechococcus elongatus PCC 7942 lacking the pipY gene, which encodes a COG0325 homolog (Labella et al., 2017;Tremiño et al., 2017). It will be very interesting to elucidate the precise function of COG0325 homologs in controlling vitamin B6 homeostasis.
The proteins YhdR, YnbB, and YwlG cannot be assigned to a specific protein family (Table 1). YhdR shares 34% overall sequence identity with an amino acid transferase from Eubacterium rectale but its role in amino acid metabolism is unknown (Table 1). Interestingly, like YaaO, YnbB, and YwlG are involved in the post-translational aminopentanol modification of the elongation factor EF-P (Witzky et al., 2018). While YwlG influences the level of the post-translational modification, YnbB seems to be required for the modification. The precise function of the proteins in controlling the activity of the elongation factor EF-P in B. subtilis needs further investigation. Moreover, it remains to be experimentally determined whether the function of YwlG depends on PLP.
The YtkP protein is a putative cysteine synthase that shares sequence similarity with the bifunctional cysteine synthase CysK and the O-acetylserine-thiol lyase MccA (Table 1). Since a cysK mccA double mutant is auxotrophic for cysteine, YtkP does not seem to be involved in cysteine biosynthesis. Thus, the function of YtkP remains elusive. The YtoP protein has been annotated as a putative glutamyl aminopeptidase because it shares about 44% overall sequence identity with PepA, a protease from Streptococcus pneumoniae that has been structurally and biochemically analyzed . Interestingly, YsdC, the paralog of YtoP (44% overall sequence identity), is annotated as an endo-1,4-β-glucanase (Table 1). Therefore, it is tempting to speculate whether YtoP is indeed involved in protein turnover. The binding of PLP to YtoP has to be experimentally validated. To conclude, B. subtilis contains several poorly characterized PLP-dependent proteins, which need to be studied in the future.

CONCLUSIONS AND FUTURE PERSPECTIVES
For a complete understanding of the vitamin B6 metabolism of B. subtilis it is crucial to identify and characterize all the proteins that require the essential co-factor to fulfill their function. However, even for well-studied model bacteria like B. subtilis the complete set of the enzymes involved in vitamin B6 metabolism and the PLP-dependent proteins remains to be identified. Several bioinformatics-driven approaches have been performed to identify and classify PLP-dependent enzymes Peracchi, 2003, 2009). Even though the PLP-dependent proteins often show low sequence similarities, using sensitive Hidden Markov Model-base sequence similarity searches PLP-dependent proteins can be identified (Yoon, 2009). However, in case the protein has a fold that is different from the known PLPdependent fold it is difficult to identify novel PLP-dependent proteins by sequence comparison. Even though structural similarity searches allowed assigning the PLP-dependent proteins to five distinct fold types (Mehta et al., 1993;Mehta and Christen, 2000;Catazaro et al., 2014), other approaches have to be pursued to uncover the full repertoire of PLP-dependent enzymes in a given organism. Indeed, mass spectrometry and biochemical approaches have been performed to identify proteins that were modified by PLP (Simon and Allison, 2009;Whittaker et al., 2015;Wu et al., 2018). As described above, a recent mass spectrometry approach identified proteins in the Gram-positive pathogen S. aureus that might depend on the B6 vitamer PLP (Hoegl et al., 2018). It will be interesting to evaluate whether the same approach will lead to the identification of novel PLPdependent proteins in B. subtilis and related bacteria. Moreover, the transport systems for the uptake and export of the B6 vitamers PN and PL have to be identified by B. subtilis. The phosphatase involved in the dephosphorylation of PNP is so far unknown (Commichau et al., 2014(Commichau et al., , 2015. Furthermore, the function of the conserved YlmE protein (YggS in E. coli) in vitamin B6 homeostasis has to be studied. Finally, it has to be elucidated how the PLP molecules are delivered to their target proteins.

SPECIALITY SECTION
PLP-Dependent Enzymes: Extraordinary Versatile Catalysts and Ideal Biotechnological Tools for the Production of Unnatural Amino Acids and Related Compounds, in Process and Industrial Biotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology.

AUTHOR CONTRIBUTIONS
BR, JR, and FC performed the database search. FC coordinated the work and wrote the manuscript with input from all authors.

FUNDING
This work was supported by the Fonds der Chemischen Industrie (to FC), the Max-Buchner-Forschungsstiftung (MBFSSt 3381 to FC), and the Deutsche Forschungsgemeinschaft (DFG grants CO 1139/1-2 and GSC 226/2 to FC and JR, respectively). This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 720776.

ACKNOWLEDGMENTS
We are grateful to members of the Commichau laboratory for fruitful comments and suggestions. We also acknowledge the foundational work on the metabolism of vitamin B6 in bacteria and other organisms that was published beforehand and cannot be fully covered because of the limit of references.