Members of a Novel Kinase Family (DUF1537) Can Recycle Toxic

Jun 13, 2016 - DUF1537 is a novel family of kinases identified by comparative genomic ...... Boyd , M. J., Logan , M. A., and Tytell , A. A. (1948) Th...
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Members of a Novel Kinase Family (DUF1537) Can Recycle Toxic Intermediates into an Essential Metabolite Jennifer J. Thiaville,† Jake Flood,‡ Svetlana Yurgel,§ Laurence Prunetti,† Mona Elbadawi-Sidhu,∥ Geoffrey Hutinet,† Farhad Forouhar,# Xinshuai Zhang,∇ Venkateswaran Ganesan,† Patrick Reddy,† Oliver Fiehn,∥,⊥ J. A. Gerlt,∇ John F. Hunt,# Shelley D. Copley,‡ and Valérie de Crécy-Lagard*,† †

Department of Microbiology and Cell Science and Genetic Institute, University of Florida, P.O. Box 110700, Gainesville, Florida 32611-0700, United States ‡ Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado United States § Dalhousie University, 6299 South St., Halifax, NS B3H 4R2, Canada ∥ West Coast Metabolomics Center, UC Davis, Davis, California, United States ⊥ King Abdulaziz University, Biochemistry Department, Jeddah, Saudi Arabia # Department of Biological Sciences, Columbia University, New York, New York, United States ∇ Institute for Genomic Biology, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States S Supporting Information *

ABSTRACT: DUF1537 is a novel family of kinases identified by comparative genomic approaches. The family is widespread and found in all sequenced plant genomes and 16% of sequenced bacterial genomes. DUF1537 is not a monofunctional family and contains subgroups that can be separated by phylogenetic and genome neighborhood context analyses. A subset of the DUF1537 proteins is strongly associated by physical clustering and gene fusion with the PdxA2 family, demonstrated here to be a functional paralog of the 4-phosphohydroxy-L-threonine dehydrogenase enzyme (PdxA), a central enzyme in the synthesis of pyridoxal-5′-phosphate (PLP) in proteobacteria. Some members of this DUF1537 subgroup phosphorylate L-4-hydroxythreonine (4HT) into 4phosphohydroxy-L-threonine (4PHT), the substrate of PdxA, in vitro and in vivo. This provides an alternative route to PLP from the toxic antimetabolite 4HT that can be directly generated from the toxic intermediate glycolaldehyde. Although the kinetic and physical clustering data indicate that these functions in PLP synthesis are not the main roles of the DUF1537-PdxA2 enzymes, genetic and physiological data suggest these side activities function has been maintained in diverse sets of organisms.

T

identifying the exact function for novel enzyme families remains difficult. Even if the general metabolic area or the chemistry involved can be elucidated, pinpointing the exact substrate is challenging. First, a substrate identified in vitro might not be the real in vivo substrate. Then, even when a substrate is identified in vivo, complications may arise due to the promiscuous nature of many enzymes.10,11 We illustrate this here and in the concomitant study,12 with the functional characterization of the DUF1537 family of proteins linking it to both carbon source catabolism and to the synthesis of the essential cofactor pyridoxal 5′-phosphate (PLP). PLP is the active form of six interconvertible B6-vitamers (pyridoxal, pyridoxine, pyridoxamine, and their 5′ phosphate derivatives). Enzymes utilizing PLP as a cofactor are found in

he lack of functional information for the vast majority of genes is a major obstacle in the study of biological systems. More than 60% of known proteins have only a vague function associated with them, and many have no function associated at all such as the members of Domain of Unknown Function or DUF families.1,2 With more than 6000 complete genome sequences (www.genomesonline.org)3 and nearly 50 000 000 protein sequences deposited into TrEMBL (http://www.ebi.ac.uk/uniprot/TrEMBLstats),4 comparative genomic analysis has emerged as a powerful tool to elucidate the roles of genes. By analyzing genomic and genome-wide experimental data, testable hypotheses can be developed regarding biological function.5,6 In order to fully characterize a novel protein family, concerted efforts are needed that combine comparative genomics, genetic analyses, and structural and biochemical characterization performed by individual groups of laboratories (see refs 7 and 8 for examples) or larger consortia (reviewed in ref 9). Despite these efforts, © XXXX American Chemical Society

Received: March 28, 2016 Accepted: June 13, 2016

A

DOI: 10.1021/acschembio.6b00279 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 1. PLP biosynthesis pathways. The steps involved in the canonical DXP-dependent PLP pathway are indicated in blue. The steps involved in the D-ribose 5-phosphate (R5P)-dependent (or DXP-independent) pathway are indicated in green. Steps performed as a result of a side reaction of an enzyme are indicated with the enzyme name in italics. The steps involved in the 4-hydroxythreonine (4HT)-dependent pathway with the proposed step for the DUF1537 kinase (STM0162) are highlighted in pink.

Figure 2. Fusion/clusters pdxA paralogs and canonical pdxA (E. coli/Salmonella) tree showing pdxA paralogs. Sequences of select canonical PdxA and DUF1537-clustered PdxA-like proteins were acquired from the SEED database and used to create a phylogenetic tree using phylogeny.fr. The UniProt ID for each sequence is listed in Table S3. The canonical PdxA proteins separate from the paralogs that cluster with DUF1537. The gene clusters of pdxA and pdxA2 are indicated by arrows. The E. coli pdxA gene neighborhood is shown for reference. The two S. typhimurium LT2 pdxA gene clusters are shown. The canonical pdxA (STM0091) cluster is the same as E. coli. The pdxA paralog (STM0163) clusters with DUF1537 (STM0162), a DeoR-like transcriptional regulator, and a KDG transporter.

threonine (4PHT) to 3-amino-1-hydroxyacetone-1-phosphate by 4-phosphohydroxy-L-threonine dehydrogenase (PdxA), followed by condensation with deoxyxylulose 5-phosphate (DXP) catalyzed by the PNP synthase (PdxJ) to make PNP. PNP is further modified to PLP by flavin mononucleotide (FMN)-dependent pyridoxine (pyridoxamine) oxidase (PdxH). This route is often called the DXP-dependent pathway (Figure 1). The second route was discovered more recently but is more widespread. It relies on the PdxS (Pdx1) and PdxT (Pdx2) enzymes that, in one step, use D-ribose 5-phosphate (R5P), glutamine, and glyceraldehyde 3-phosphate to directly make

all organisms, catalyzing a variety of reactions such as transaminations, decarboxylations, racemizations, and β- and γ- carbon elimination/replacements, and are mostly associated with amino acid metabolism (http://bioinformatics.unipr.it/ B6db).13 Most bacteria, plants, and fungi make PLP de novo.14 Although the quest for PLP synthesis pathways started in the 1960s and 1970s, it was only recently that these were elucidated in most major model organisms14−16 (Figure 1). One route, discovered first in E. coli, but restricted to proteobacteria and bacteroides, relies on the oxidation of 4-phosphohydroxy-LB

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TphB, involved in terephthalate degradation.27 Additionally, other genes that strongly cluster with pdxA2 and DUF1537 genes encode a putative transcriptional regulator involved in sugar metabolism and a 2-keto-3-deoxygluconate permease (Figure 2), suggesting the role of PdxA2 and DUF1537 may be in carbon source metabolism rather than PLP biosynthesis. To genetically test whether members of the PdxA2 family harbored 4PHT dehydrogenase activity, we attempted to complement the PLP auxotrophy of an E. coli pdxA::kan mutant24 with pdxA2 from S. typhimurium LT2 (gene locus STM0163). When pdxA2 was expressed from a PTET promoter on the low copy vector pBY279,28 it complemented the PLP auxotrophy of E. coli pdxA::kan (Figure 3A).

PLP in what is called the R5P-dependent or the DXPindependent pathway (Figure 1). A glycolaldehyde-dependent route was previously thought to be the major pathway for PLP synthesis in E. coli through the formation of 4-hydroxythreonine (4HT;17 Figure 1). This was discarded when the physiological DXP-dependent route was established in this organism18 (Figure 1). The glycolaldehyde/ 4HT route was found to be a serendipitous pathway in E. coli using side reactions of enzymes from other pathways, e.g., threonine aldolase (LtaE) and the homoserine kinase (ThrB;19 Figure 1). The relevance of the glycolaldehyde route as a physiological source of PLP remains, however, a possibility in other organisms such as Sinorhizobium meliloti, where labeling studies suggest that both the DXP-dependent and the 4HTdependent pathways are active.20−22 By exploring the network of genes associated with PLP synthesis enzymes by comparative genomics,6 we found that a subset of DUF1537 family genes was strongly associated with pdxA paralog genes. Members of the DUF1537 family are grouped based only on sequence similarity in the Pfam database.2 Here and in the complementary study,12 we demonstrate that DUF1537 proteins comprise a novel family of kinases, involved in the catabolism of C4 carboxylic acids.12 We show here that a subset of these proteins, encoded as a fusion with or adjacent to pdxA paralogs, are promiscuous and will phosphorylate 4HT to the PLP pathway intermediate 4PHT, both in vivo and in vitro.

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RESULTS AND DISCUSSION Comparative Genomic Analyses of the DUF1537 Family. When systematically exploring functional associations between genes of unknown function and PLP synthesis genes, we found that genes encoding members of the DUF1537 family were strongly associated with genes annotated as pdxA (Figure 2), all encoding members of the PF04166 Pfam family. In at least 32 organisms identified in the Pfam and PubSEED databases, the DUF1537 family gene was fused to the annotated pdxA gene, resulting in a fusion protein. In most other cases, the genes were adjacent or encoded nearby one another (see the DUF1537-pdxA2 subsystem). Upon closer observation, it was found that the DUF1537-clustered gene annotated as pdxA was not the canonical pdxA. Indeed, when the DUF1537-pdxA genes are present in an organism that synthesizes PLP through the DXP-dependent pathway (as indicated by the presence of the pdxJ gene; Figure 1), there is always a second copy of pdxA with greater similarity to the canonical pdxA. A phylogenetic analysis of the PdxA family shows that the DUF1537-linked PdxA is a paralog of the canonical PdxA (Figure 2). We henceforth refer to this paralog as PdxA2. In Salmonella enterica sv. Typhimurium LT2, for example, the DUF1537 family member STM0162 clusters with pdxA2 (STM0163), whereas the canonical pdxA (STM0091) clusters with surA and ksgA, like its E. coli K12 counterpart23,24 (Figure 2). PdxA2/STM0163 Is a Functional 4-Phosphohydroxythreonine Dehydrogenase. Out of the 378 known organisms that harbor clustered DUF1537-pdxA2 genes, 60% (230) have a canonical pdxA gene and 30% (112) harbor a pdxS gene, indicative of the DXP-independent route to PLP. Many of the remaining 10% are known vitamin B6 auxotrophs, such as Clostridium spp.25 and Lactobacilli.26 It was therefore possible that the function of PdxA2 is unrelated to PLP synthesis, as already observed with another PdxA paralog,

Figure 3. Complementation of E. coli vitamin B6 auxotrophs by pdxA2 and DUF1537 of S. Typhimurium LT2. (A) E. coli pdxA::kan carrying pVG05 (pPTET::pdxAST), pJJT37 (pPTET::pdxA2ST), or pBY279 (empty vector) were streaked on M9 agar with 0.2% glucose, chloramphenicol, and anhydrotetracline with or without pyridoxine. (B) Dilution drops of E. coli ΔpdxB::kan, E. coli ΔpdxB ΔthrB::kan, and E. coli ΔthrB::kan carrying either pJJT50 (pPTET::STM0162) or pBY279 (pPTET::empty), see Table 2 legend for growth conditions.

To confirm that PdxA2 is a functional 4PHT dehydrogenase, we purified a recombinant PdxA2ST from E. coli (Figure S1) and measured the 4PHT dehydrogenase activity in vitro (Table 1). The PdxA2ST had 4PHT dehydrogenase activity in vitro with a kcat/KM of 8.3 ± 2.5 × 103 M−1 s−1, just an order of magnitude lower than the canonical E. coli PdxA with a kcat/KM of 1.2 ± 0.4 x105 M−1 s−1. Both the genetic and chemical evidence shows that the PdxA2 has retained 4PHT dehydrogenase activity even if the codistribution analyses with canonical PLP synthesis genes and the physical clustering evidence suggested otherwise. Genome Context and Structural Analysis Suggest a Kinase Role for DUF1357 Members. The DUF1537 family is not monophyletic, and many organisms contain paralogs (see DUF1537-pdxA2 subsystem). For example, S. meliloti contains three members of the DUF1537 family (Figure 4). These paralogs can be separated based on gene neighborhoods. One of the DUF1537 subgroups clustering with pdxA2 (discussed above) that includes STM0162 also clusters with a transcriptional regulator, and a 2-keto-3-deoxygluconate permease-like protein (Figure 4). In some bacteria, like S. meliloti and C

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ACS Chemical Biology Table 1. Kinetic Data for PdxA, PdxA2, and STM0162 protein

substrate

PdxA PdxA2 (STM0163)b STM0162b

4PHT 4PHT D-threonate 4-HT

a

a

kcat (s−1) 0.81 0.06 22 4.0

± ± ± ±

0.17 0.0088 1.3 0.1

KM (μM)

kcat/KM (M−1 s−1)

± ± ± ±

1.15 ± 0.35 × 105 8.25 ± 2.49 × 103 7.5 × 104 1.1 × 103

7.03 7.24 0.29 3.8

4.73 3.52 0.05 0.5

pdxA gene cloned from E. coli BW25113. bSTM0163 (pdxA2) and STM0162 (DUF1537) genes cloned from S. typhimurium LT2.

Figure 4. Subfamilies of the DUF1537 family. The UniProt ID for each sequence is listed in Table S3. Gene clusters for representative DUF1537 genes discussed in this study are shown to the right. DUF1537 genes are shown in red. The other colors correlate to related functions. lysR, lysR-like transcriptional regulator; gntP, gluconate permease; SDR, nucleoside-diphosphate-sugar epimerase/short-chain dehydrogenases/reductase; ygbM, hydroxypyruvate isomerase; ygbL, putative class II aldolase; ygbJ, D-beta-hydroxybutyrate dehydrogenase; ygbI, deoR-like transcriptional regulator; marR, marR-like transcriptional regulator; IS5, insertion element; lacI, lacI-like transcriptional regulator; xylF, similar to sugar ABC transporter; RLP, RUBISCO-like protein; KDG perm, 2-keto-3-deoxygluconate permease; dapA, dihydrodipicolinate synthase family; ABC, ABC transporter proteins; adh, alcohol dehydrogenase.

Pectobacterium atrosepticum, this DUF1537-pdxA2 cluster is expanded to include a predicted alcohol dehydrogenase, a sialidase, a dihydrodipicolinate synthase, and an ABC-type nickel/dipeptide/oligopeptide-like import system system (Figure 4 and Figure S5). A second subgroup consists of the very conserved DUF1537 cluster of seven genes (ygbIJKLMN in E. coli and S. typhimurium LT2) most certainly involved in carbon source utilization as these genes are predicted to encode a DeoR-family transcriptional regulator (ygbI), 3-hydroxyisobutyrate dehydrogenase (ygbJ), DUF1537 (ygbK), a class II aldolase (ygbL), hydroxypyruvate isomerase (ygbM), and a 2keto-3-deoxygluconate (KDG) permease homologue (ygbN) (Figure 4). A third DUF1537 subgroup clusters with a rubiscolike protein (RLP), a KDG transporter, and GntR-type transcriptional regulator, again suggestive of carbon source metabolism. As with the DUF1537-pdxA2 group, each of these subgroups has variations of the other genes encoded within each neighborhood context, suggesting that even within our three broadly defined subgroups the DUF1537 proteins may not be monofunctional. If these clusters encode genes involved either in the alternative PLP synthesis route or in the catabolism of an unknown sugar, an obvious missing component is a kinase. A common step in sugar catabolism is phosphorylation,29 and as discussed above, the 4HT route to PLP requires a kinase. The three-dimensional structure of two members of the DUF1537 family, both from the YgbK subgroup (PDB IDs: 3DQQ and 1YZY), had been solved through the structural genomics effort

at the time of our analysis. No structural similarity to any known enzyme was observed. However, in the fold analysis performed in SCOP,30 the YgbK family was found to be similar to P-loop kinases, but lacking the P-loop motif. In light of this similarity, we purified STM0162 (Figure S1) and found that ATP induced a change in thermal stability of the purified STM0162 (Figure S2) as indicated by an increase in midpoint temperature (56 °C in the absence of ATP vs 58 °C in the presence of ATP). Titration in the presence of 5 mM Mg-ATPγ-S showed roughly the same midpoint temperature as in the presence of 5 mM Mg-ADP (data not shown). Titrations of the protein in the presence or absence of 5 mM MgCl2 showed no significant shift in midpoint temperature, while the absence of Mg2+ from the titration conducted in the presence of 5 mM Mg-ATP-γ-S resulted in a reduction of less than 1° in midpoint temperature (data not shown). The stabilization of the protein by ATP is consistent with the possibility that the protein is a kinase. The combination of a potential kinase function and an association with PdxA2, active as a 4PHT dehydrogenase, led us to propose that the members of the DUF1537 family that cluster with pdxA2 might catalyze the phosphorylation of 4HT into the PdxA2 substrate 4PHT. STM0162 Has 4-Hydroxythreonine Kinase Activity in Vivo and in Vitro. Because the homoserine kinase ThrB can phosphorylate 4HT as a side reaction,18,31 thrB is required for an E. coli strain lacking the 4-phosphoerythronate dehydrogenase PdxB to grow when supplemented with 4HT or D

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ACS Chemical Biology glycolaldehyde.18,19 This observation provided a phenotypic test for the predicted 4HT kinase activity of the pdxA2-linked DUF1537 encoded by STM0162 in S. typhimurium LT2 (Table 2 and Figure 3B). As shown in Figure 3B, expressing STM0162

DUF1537. Indeed, as the corresponding study by Zhang et al.12 demonstrates, the DUF1537 enzyme phosphorylates Dthreonate in vitro with a kcat/KM of 7.5 × 10 4 M−1 s−1 (Table 1). To test whether the kinase activity was responsible for the observed complementation of E. coli ΔpdxB ΔthrB::kan, residues predicted to bind ATP or the substrate were chosen for site-directed mutagenesis. In the study by Zhang et al.,11 the crystal structure of the DUF1537 (PDB: 5DMH) of Ralstonia eutropha bound to ADP revealed that a conserved Gly414 interacted with the ADP molecule. We mutated the corresponding site of the S. typhimurium LT2 STM0162 and confirmed that a G401A change resulted in a loss of the ability to complement the PLP auxotrophy of E. coli ΔpdxB ΔthrB::kan (Table 3).

Table 2. Ability of DUF1537ST (STM0162) to Complement Different PLP Auxotrophsa empty vector supplement added:

PN

GA

E. coli BW25113 ΔpdxB ΔpdxB ΔthrB::kan ΔpdxB ΔltaE::kan ΔpdxB ΔthrB ΔltaE::kan ΔpdxB ΔpdxJ::kan ΔpdxB ΔthrB ΔpdxJ::kan ΔpdxB pdxA::kan ΔpdxB ΔthrB pdxA::kan

+ + + + + + + + +

+ + − − − − − − −

DUF1537ST + − − − − − − − −

PN

GA

+ + + + + + + + +

+ + + + − − − − −

+ + + − − − − − −

Table 3. Complementation of the pdxB thrB Vitamin B6 Auxotrophy by Different DUF1537 Genesa

a

All strains were grown on M9 agar, glucose with L-threonine, chloramphenicol, and anhydrotetracycline. PN = 500 ng/μL pyridoxine; GA = 200 nM glycolaldehyde; + = growth, − = no growth after 40 h at 37 °C.

plasmid

organism

pJJT79

Actinobacillus succinogenes 130Z Nostoc punctiforme PCC 73102 Pectobacterium atrosepticum SCRI1043 Prochlorococcus marinus subsp. marinus str. CCMP1375 S. typhimurium LT2 Mannheimia hemolytica PHL213 Escherichia coli MG1655 Sinorhizobium meliloti 1021 Sinorhizobium meliloti 1021 S. typhimurium LT2 DUF1537 G401A

pJJT82

under the control of a PTET promoter allowed the growth of E. coli ΔpdxB ΔthrB::kan in the absence of exogenous vitamin B6 when anhydrotetracycline was added. In the serendipitous glycolaldehyde-dependent PLP pathway described by Kim et al.,19 4HT is formed by condensation of glycolaldehyde and glycine by the low-specificity threonine aldolase LtaE. STM0162 was unable to complement the PLP auxotrophy of E. coli ΔpdxB ΔthrB ΔltaE::kan (Table 2), suggesting that in E. coli, STM0162 is phosphorylating the 4HT produced from glycolaldehyde by LtaE. It is known that, in E. coli, the 4HT-dependent route requires PdxA and PdxJ.17 We reproduced this observation (Table 2) and found these two genes were also required for the STM0162-mediated PLP production (Table 2). We also showed that the STM0162-mediated complementation did not require the action of the phosphohydroxythreonine aminotransferase (SerC/PdxF) to produce 4PHT from 2-oxo3-hydroxy-4-phosphobutanoate as STM0162 was able to complement the PLP auxotrophy of E. coli ΔthrB ΔpdxF::kan (data not shown) when supplemented with threonine, serine, and glycolaldehyde on an M9-glucose medium. Hence, genetic evidence suggests that DUF1537 is capable of the phosphorylation of 4HT derived from glycolaldehyde to 4PHT, the substrate of PdxA, bypassing the need for PdxB and PdxF enzymes. We set out to confirm this by mass spectrometry. 4PHT is detected in E. coli ΔpdxB ΔthrB pdxA::kan expressing STM0162 when grown in an M9-glucose medium supplied with pyridoxine, threonine, and glycolaldehyde. When STM0162 is not present, 4PHT is not detected, although 4HT is still present, confirming that DUF1537 phosphorylates 4HT to 4PHT in vivo (Figure S3). To further validate the in vivo results, we tested the 4HT kinase activity in vitro. STM0162 was purified from E. coli as described in the Methods section and was found to phosphorylate 4HT in vitro with a kcat/KM of 1.1 × 103 M−1 s−1 (Table 1), the same order of magnitude as the promiscuous activity of ThrB, which phosphorylates 4HT with a kcat/KM of 4.8 ± 0.6 × 103 M−1 s−1, as described in ref 19. Similar to ThrB, 4HT kinase activity is likely a promiscuous activity of

pJJT83 pJJT130 pJJT50 pJJT85 pJJT57 pJJT162 pJJT163 pJJT190

cluster/ group

-

4HT

GA

PN

pdxA-linked

+

+

+

+

DUF1537/ other pdxA-linked

−

−

−

+

−

−

+

+

DUF1537/ other

−

−

−

+

pdxA-linked pdxA-linked

+ −

+ −

+ −

+ +

ygbK-like

−

−

−

+

pdxA-linked

−

−

+

+

RLP-linked

−

−

−

+

pdxA-linked

−

−

−

+

a

All strains were grown on M9 agar with glucose, L-threonine, chloramphenicol, and anhydrotetracycline. - = no additive; 4HT = 100 nM 4-hydroxythreonine; GA = 200 μM glycolaldehyde; PN = 500 ng/ μL pyridoxine.

Physiological Relevance of a 4HT-Dependent PLP Synthesis in Bacteria. S. typhimurium LT2 has both an intact DXP-dependent PLP synthesis pathway with the pdxA gene and a DUF1537-pdxA2 cluster (Figure 2). We first determined whether the presence of any of the pdxA genes was sufficient to drive the DXP-dependent pathway. Unlike the E. coli pdxA mutant,24 the S. typhimurium LT2 ΔpdxA::kan strain grew, albeit slowly, in the absence of a PLP source, indicating that it was not a complete vitamin B6 auxotroph. The growth of the ΔpdxA::kan strain was improved slightly by the addition of glycolaldehyde, and even more with 4HT (Figure 5B). Deletion of pdxA2 alone had no effect on growth in M9-glucose (Figure 5A); however, deleting pdxA2 in the ΔpdxA::kan background eliminated all growth in the absence of PLP (Figure 5A). In S. typhimurium LT2, the 4HT-dependent route seems to be capable of functioning as a back-up synthesis route to PLP. However, as glycolaldehyde does not significantly increase growth of S. typhimurium LT2 ΔpdxA::kan like exogenous 4HT E

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Figure 5. Growth of S. typhimurium LT2 pdxA and pdxA2 mutants. (A) Growth curves of wild-type S. typhimurium LT2, VDC9536 (ΔpdxA::kan), VDC9538 (ΔpdxA2::cat), and VDC9852 (ΔpdxA::kan ΔpdxA2::cat) in M9-glucose at 37 °C. (B) Growth curves of VDC9536 (ΔpdxA::kan) and VDC9852 (ΔpdxA::kan ΔpdxA2::cat) in M9-glucose supplemented with either pyridoxine (PN), glycolaldehyde (GA), 4-hydroxythreonine (4HT), or no additive. (C) S. typhimurium LT2, VDC9536 (ΔpdxA::kan), VDC9538 (ΔpdxA2::cat), and VDC9852 (ΔpdxA::kan ΔpdxA2::cat) were streaked onto M9-agar with glucose supplemented with pyridoxine, no additive, or glycolaldehyde and incubated at 37 °C for 24 h.

(Figure 5), the question of the potential source of 4HT in S. typhimurium LT2 remains unanswered. The plant symbiont S. meliloti presents an interesting case whereby there are three pdxA paralogs and three DUF1537 paralogs (Figures 2 and 4 and Supporting Information Results). The canonical pdxA (SMc00580, called here pdxA1), is likely to encode the major 4PHT dehydrogenase as deletion of pdxA1 substantially, but not completely, decreased the growth of S. meliloti in the absence of pyridoxine (Figure S4). The partial growth suggests one or both of the other PdxA paralogs maintain some side 4PHT dehydrogenase activity. S. meliloti uses pdxR, a gene not orthologous to pdxB, for the dehydrogenation of 4-phosphoerythronate.21 Loss of the pdxR gene causes a pyridoxine auxotrophy that is partially restored by 4HT, but not glycolaldehydee (Figure S4), suggesting that in S. meliloti, like in E. coli or S. typhimurium LT2, PLP can be synthesized from 4HT independently of PdxR/PdxB activity. The fact that adding glycolaldehyde did not restore growth is discussed in the Supporting Information Results. There are several arguments against a primary role of the PdxA2/DUF1537 pathway in PLP synthesis. First, organisms that are not PLP auxotrophs and contain the PdxA2/DUF1537 genes always contain one of the two known PLP pathways as predicted from the presence of PdxJ or Pdx1/Pdx2 (Figure 1 and DUF1537-pdxA2 SEED subsystem). More importantly, the concomitant study by Zhang et al.11 shows that STM0162 phosphorylates D-threonate and D-erythronate in vitro more efficiently than 4HT11 and that the corresponding products Dthreonate-4-phosphate and D-erythronate-4-phosphate, then undergo oxidative decarboxylation by PdxA2 (STM0163) to produce dihydroxyacetone phosphate (DHAP) and CO2. In addition, Zhang et al.11 show that D-threonate is utilized as a carbon source by S. typhimurium LT2 through the DUF1537PdxA2 pathway. As PLP synthesis is certainly not the main function of the PdxA2 and DUF1537 enzymes, we propose they comprise an

alternative pathway to make PLP out of a toxic metabolite, 4HT. 4HT can be formed endogenously by several routes such as through the LtaE side reaction discussed above19 or through the hydroxylation of threonine by a novel family of dioxygenases32 and prior to the discovery of the DXPdependent pathway in Proteobacteria, 4HT was thought to be an intermediate in PLP biosynthesis.17 4HT was coined as an “antimetabolite” when isolated from an unidentified Streptomycetes and shown to inhibit E. coli growth.33 This growth inhibition was relieved by the addition of L-serine or Lthreonine,34 and deregulation of the expression of the homthrB-thrC operon renders B. subtilis resistant to 4HT,35 confirming that 4HT interferes with threonine biosynthesis. The phosphorylation of 4HT by ThrB into 4PHT, a known inhibitor of threonine synthase (ThrC),31,36−38 is most likely a cause of growth inhibition. The side reactions of the DUF1537 and PdxA2 enzymes may serve as an elegant solution by the bacteria to deal with the toxicity of 4HT by converting it into a useful product. Functional Diversity of the DUF1537 Family. As DUF1537 proteins that cluster with PdxA2 represent only a fraction of the DUF1537 family, we tested other members of the DUF1537 family for in vivo 4HT kinase activity (Figure S5 and Table 3). Using the genetic test described above, we found that only members of the DUF1537 subfamily that clustered with pdxA2 complemented the PLP auxotrophy of a strain lacking pdxB and thrB PLP (Table 3). The Actinobacillus succinogenes DUF1537 (Asuc_0446) complements the PLP auxotrophy in minimal medium with or without 4HT or glycolaldehyde (Table 3). Both the S. meliloti DUF1537 gene (SMb20149) and the Pectobacterium atrosepticum DUF1537 gene (ECA3761) linked to pdxA2 complement the PLP auxotrophy of E. coli ΔpdxB ΔthrB::kan only in the presence of glycolaldehyde, not 4HT. These DUF1537 genes are in expanded gene clusters compared to those of S. typhimurium LT2 and A. succinogenes (Figure 4 and Figure S5). These DUF1537 enzymes may be phosphorylating another substrate F

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Foundation (NSF; grant MCB-1153413 to V.d.C.-L. and MCB 1153491 to O.F.) and the National Institutes of Health (NIH 1S10RR031630-01 to O.F., NIHNIH U54GM093342 to J.A.G., and NIH GM083285-05 to S.D.C.).

derived from glycolaldehyde that can feed into the PLP pathway. The members of DUF1537 that are not associated with pdxA2, and even one that is associated with pdxA2, have lost 4HT kinase activity (Figure S5 and Table 3), showing that this activity is not just a side reaction of the DUF1537 family at large. Conclusion. Our discovery that a subset of the DUF1537 family phosphorylates 4HT is a tribute to the power of using comparative genomic analyses in combination with experimental validation. We correctly predicted a novel kinase family using these methods. It also shows the difficulty and potential pitfalls in calling a definitive gene function. Indeed, although we have both in vivo and in vitro evidence that some DUF1537 proteins phosphorylate 4HT, this does not seem to be the main physiological function of this enzyme family. The work presented here and in the Zhang study12 also emphasizes the difficulty of annotating families with paralogs with different substrate specificities and shows that these require a combination of phylogenetic, genome context, and experimental evidence to be correctly annotated.

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METHODS

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Bioinformatic Analyses. The BLAST tools39 and resources at NCBI (http://www.ncbi.nlm.nih.gov/) were routinely used. Multiple sequence alignments were built using Clustal Omega4 or Multalin.40 Protein domain analysis was performed using the Pfam database tools2 and the Conserved Domain Database (CDD) at NCBI.41 Analyses of phylogenetic distribution and physical clustering were performed in the SEED database.42 Results are available in the “DUF1537-pdxA2” subsystem (http://pubseed.theseed.org/SubsysEditor.cgi?page= ShowSubsystem&subsystem=DUF1537-pdxA2) on the public SEED server (http://pubseed.theseed.org/SubsysEditor.cgi). Physical clustering was analyzed with the SEED subsystem coloring tool or the SeedViewer Compare Regions tool.42 The protein association network analysis was performed on the String database (string-db.org).43 The Interactive Tree of Life v2 (ITOL) platform was used to build the gene distribution trees (http://itol.embl.de/index.shtml).44 Phylogenetic trees were constructed using phylogeny.fr.45 Structures were visualized with the Protein Data Bank (PDB) tools (www.rcsb.org).46 Genetic and Biochemical Experiments. All strain and plasmid constructions as well as the methods for genetic, biochemical, and analytical analyses as described in the Supporting Information Methods. S Supporting Information *

This material is available free of charge via the Internet. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00279. (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (352) 392 9416. Fax: (352) 392 5922. E-mail: vcrecy@ ufl.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank S. Shanker and the staff at UF ICBR for Sanger DNA sequencing. We thank M. Winkler for numerous strains, Ian D. Spenser for inspiration, and B. El Yacoubi for insightful discussions. This work was funded by the U.S. National Science G

DOI: 10.1021/acschembio.6b00279 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

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