Mutational and functional analyses of substrate binding and catalysis of the Listeria monocytogenes EutT ATP:Co(I)rrinoid adenosyltransferase
ATP:Co(I)rrinoid adenosyltransferases (ACATs) catalyze the transfer of the adenosyl moiety from co-substrate ATP to a corrinoid substrate. ACATs are grouped into three families, namely CobA, PduO, and EutT. The EutT family of enzymes is further divided into two classes, depending on whether they require a divalent metal ion for activity (Class I and Class II). To date, a structure has not been elucidated for either class of the EutT family of ACATs. In this work, results of bioinformatics analyses revealed several conserved residues between the C-terminus of EutT homologues and the structurally characterized Lactobacillus reuteri PduO (LrPduO) homologue. In LrPduO, these residues are associated with ATP-binding and formation of an inter-subunit salt bridge. These residues were substituted, and in vivo and in vitro data support the conclusion that the equivalent residues in the metal-free (i.e., Class II) Listeria monocytogenes EutT (LmEutT) enzyme affect ATP binding. Results of in vivo and in vitro analyses of LmEutT variants with substitutions at phenylalanine and tryptophan residues revealed that replacement of the phenylalanine residue at position 72 affects access to the substrate binding site and replacement of a tryptophan residue at position 238 affected binding of the Cbl substrate to the active site. Unlike the PduO family of ACATs, a single phenylalanine residue is not responsible for displacement of the \xce\xb1-ligand. Together, these data suggest that while EutT enzymes share a conserved ATP-binding motif and an inter-subunit salt bridge with PduO family ACATs, Class II EutT family ACATs utilize an unidentified mechanism for Cbl lower ligand displacement and reduction that is different from that of PduO and CobA family ACATs.
INTRODUCTION.<!>Bioinformatics analysis.<!>Site-directed mutagenesis.<!>Strains and growth media.<!>Protein purification.<!>Western blotting.<!>In vitro corrinoid adenosyltransferase activity assays.<!>Spectroscopic analysis.<!>Structural modeling.<!>EutT shares C-terminal sequence homology with PduO.<!>In vivo assessment of nucleotide binding and salt bridge variants.<!>In vitro activity of putative EutT ATP-binding variants.<!>In vitro analysis of the putative inter-subunit, salt-bridge variants.<!>Oligomerization state of the inter-subunit, salt-bridge variants.<!>Screening for residues involved in 4C-Co(II)Cbl formation.<!>LmEutTF72 and LmEutTW238 contribute to 5C-Co(II)Cbl active-site access and affinity, respectively.<!>Spectroscopic analysis of the F72X and W238H variants.<!>Modeling LmEutT and SeEutT sequences to the reported LrPduO structure (PDB code 3CI1) emphasizes similarities in the conserved regions.<!>EutT and PduO ACATs share conserved residues that bind ATP.<!>EutT ACATs may form an inter-subunit salt bridge.<!>The mechanism of base displacement in LmEutT is unique among ACAT classes.<!>CONCLUSIONS.
<p>Cobamides (Cbas) are structurally complex cyclic tetrapyrroles produced exclusively by some prokaryotes and required by organisms from all domains of life.1 The best-studied cobamide is cobalamin (Cbl), whose vitamin form is cyanocobalamin (CNCbl), and biologically active form is adenosylcobalamin (AdoCbl). AdoCbl is required by several enzymes, such as methylmalonyl-CoA mutase (EC 5.4.99.2), class II ribonucleotide reductase (EC 1.17.4.2), diol dehydratase EC 4.2.1.28), and ethanolamine ammonia-lyase (EAL, EC 4.3.1.7).2–4
Salmonella enterica and several other prokaryotes produce EAL in order to use ethanolamine as a source of carbon, nitrogen, and energy.5 EAL is encoded by the eutBC genes in the ethanolamine utilization (eut) operon,6 which in S. enterica is comprised of 16 genes (eutSPQTDMNEJGHABCLK) followed by an independently expressed regulator (eutR).7 The eutT gene encodes an ATP:Co(I)rrinoid adenosyltransferase (ACAT) that generates the AdoCbl coenzyme used by EAL.8, 9 In general, ACATs facilitate the adenosylation of corrinoids via the displacement of the lower axial ligand (α-ligand) of cobamides (Cbas) generating a four-coordinate (4C) Co(II)corrinoid intermediate that can be reduced by free or protein-bound dihydroflavins (Fig. 1).10, 11</p><p>Three families of ACATs have been described, namely CobA, PduO, and EutT, all of which have been genetically well characterized in S. enterica. CobA is considered the housekeeping ACAT of S. enterica, and is involved in de novo cobalamin biosynthesis, adenosylation of incomplete corrinoids, transcriptional activation of the eut operon, and post-transcriptional regulation of the gene encoding the outer membrane corrinoid transporter, btuB.8, 12, 13 Structural and spectroscopic analysis of S. enterica CobA demonstrated that the enzyme binds 4C-Co(II)Cbl, prior to its reduction to Co(I)Cbl and subsequent transfer of the adenosyl (Ado) moiety of ATP to the Co ion of the corrinoid substrate.14, 15 In CobA, five-coordinate (5C)-Co(II)Cbl is converted to 4C-Co(II)Cbl by displacement of the α-ligand of the corrinoid by residues F91 and W93, which are located within a disordered loop that swings into place under the corrin ring upon binding of 5C-Co(II)Cbl to the active site. The ATP-binding site of CobA is comprised of an inverted P-loop that positions the C-5' of ATP proximal to Co(I) allowing the formation of the Co–C organometallic bond.</p><p>In S. enterica, PduO is important for the metabolism of 1,2-propanediol; however, many organisms (including humans) only encode PduO-type ACATs and utilize these enzymes in a 1,2-propanediol-independent manner.16–18 Of note, humans encode a PduO homologue that delivers AdoCbl to methylmalonyl-CoA mutase (MCM).19–21 In all homologues where structural information has been elucidated, PduO forms a trimer.22–24 In the PduO trimer, each active site is located at the interface of two subunits, and residues from both subunits contribute to substrate binding. Additionally, the well-conserved R128 residue from the Lactobacillus reuteri PduO homologue (LrPduO), which is essential for adenosylation activity, appears to form an inter-subunit salt bridge with residue D35.16</p><p>In S. enterica, EutT is encoded as a member of the eut operon and plays an important role in the catabolism of ethanolamine. To date, X-ray structural data of members of the EutT family enzymes have not been reported; however, in vivo and in vitro characterization of EutT homologues have provided insights into the mechanism of this family of ACATs.25–30 EutT family ACATs can be divided into two classes, one class of enzymes that binds and requires Fe(II) or other divalent transition metal ions for activity (Class I),27, 31 and another class that lacks a metal-binding site and does not require Fe(II) for activity (Class II).26, 28
S. enterica EutT (SeEutT), the best studied Class I EutT, binds Fe(II) using a conserved HX11CCXXC motif at the dimer interface.27 Spectroscopic analysis of SeEutT, where the Fe(II) ion was replaced with Co(II), demonstrated that the metal is tetrahedrally coordinated by four cysteines, two from each subunit.31 Interestingly, SeEutT initially generates a 5C-Co(II)Cbl intermediate, whereas the Class II EutT from Listeria monocytogenes (LmEutT) binds 4C-Co(II)Cbl, similar to PduO and CobA family enzymes.28
LmEutT also forms a tetramer, a unique oligomeric state in all ACATs studied to date.26</p><p>In this work, we describe the results of a directed mutagenic analysis of the Class II EutT ACAT from Listeria monocytogenes (LmEutT). This analysis was guided by the finding that primary sequences of PduO family ACATs align with EutT family ACATs, except EutT homologues have a ~70 aa N-terminal extension that PduO homologues lack. The results suggest that EutT family ACATs bind the ATP substrate and form an inter-subunit salt bridge similarly to LrPduO.</p><!><p>Amino acid sequences of crystallized PduO homologues, EutT homologues from both classes that demonstrated activity in vitro, and the human adenosyltransferase (hATR) were aligned in Geneious (version 8.1.9) using the ClustalW BLOSUM cost matrix, with a gap open cost = 9 and gap extension = 1. This alignment was annotated in ESPript 3.0 incorporating the secondary structure of Lactobacillus reuteri PduO, with an identity cutoff of 0.7.</p><!><p>Plasmids pEUT140 and pEUT159 encoding LmEutT (UniProt accession code A0A0H3GFM5)26 were mutagenized using the QuikChange II site-directed mutagenesis protocol (Agilent), with the changes noted: 150 ng of template DNA (pEUT140 or pEUT159) was used for the amplification, and the DpnI digestion was incubated at 37 °C for six hours. Primers used for the amplification of each variant are listed in Table S2. Candidate plasmids were sequenced to confirm the presence of the correct mutation. A list of the plasmids used in this work can be found in Table S1.</p><!><p>For protein expression, pTEV18 32 vector-based plasmids encoding LmEutT variants were transformed into BL21-CodonPlus cells (Agilent) and grown as previously described.26 For analysis of ACAT activity in vivo, pTAC-85 33 vector-based plasmids encoding LmEutT variants were transformed into the reporter strain JE22174. Strains were grown in 1 mL of nutrient broth (NB, Difco) (8 g/L) containing NaCl (5 g/L) and were incubated overnight at 37 °C. These cultures were subcultured into minimal medium containing lactose as the sole source of carbon and energy, as described elsewhere.26</p><!><p>Purification of LmEutT variants and analysis of their purity was performed as described elsewhere26 If variants were unstable, as in the case of LmEutTR200D and LmEutTD110R R200D, some protein precipitated and was removed by centrifugation at 2,000 × g for 10 minutes at 4 ºC in an Eppendorf Centrifuge 5810R centrifuge. The remaining soluble protein was decanted and filtered through a 0.45-μm filter (VWR) to remove particulates before storage at −80 ºC until used.</p><!><p>Strains were grown on 5 mL NCE medium 34 containing Wolfe's trace minerals 35 and MgSO4 (1 mM) supplemented with lactose (22 mM), glycerol (22 mM), ethanolamine (1 mM), cyanocobalamin (CNCbl, 25 nM), ampicillin (20 μM) and isopropyl β-D-thiogalatopyranoside (IPTG, 10 μM). Strains were grown to an optical density (OD) of 0.6–0.8 at 600 nm. Cells were pelleted by centrifugation at 4,000 × g using an Beckman Avanti™ refrigerated centrifuge furnished with a JA25.50 rotor. Cell pellets were stored at –80 °C until used. Cell pellets were resuspended in 300 μL of B-PER™ reagent (Thermo Fisher Scientific) and insoluble debris was centrifuged at 16,000 × g for 5 minutes on a Eppendorf Centrifuge Model 5425. Cell lysates were quantified with the Pierce BCA protein assay kit (Thermo Fisher Scientific) and 100 μg of each lysate was loaded into two 15% polyacrylamide SDS-PAGE gels 36 in duplicate. One gel was loaded with a Precision Plus Protein Standards (Bio-Rad Laboratories) and the other gel was loaded with SuperSignal™ Molecular Weight Protein Ladder (Thermo Fisher). A constant voltage of 200 V was applied for 50 min to separate the proteins in each lysate by size on the gel. One gel was stained with Coommassie Brilliant R dye to visualize the protein content of each sample. The other gel was transferred to a PVDF membrane using the Bio-Rad Laboratories Trans-Blot Turbo transfer system. After blocking the membrane with 1X Phosphate-Buffered Saline with 0.1% (v/v) Tween (PBST) solution and 0.5% (w/v) non-fat milk for one hour, the membrane was incubated with the same blocking buffer with rabbit polyclonal antibodies against LmEutT (Envigo, PA) that were precleared with lysate from the background strain harboring the empty cloning vector grown under the same conditions; the antibody dilution used was 1:500 for the primary antibody. The membrane was washed thrice with PBST, 10 minutes per wash, and a second hour-long incubation was performed with blocking buffer containing goat anti-rabbit IgG, horseradish peroxidase conjugate (Sigma) at a 1:10,000 dilution. The membrane was again washed thrice with PBST before applying 10 mL of SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) and incubating for 10 min. The blot was imaged with a UVP ChemStudio (analytikjena) with an exposure time of 10 min.</p><!><p>Adenosyltransferase activity assays were prepared as described elsewhere with minor changes.26 Enzyme assays had an enzyme concentration of 400 nM for the determination of the kinetic parameters, and these concentrations were quantified using a NanoDrop 1000 spectrophotometer with the appropriate molecular weight and extinction coefficient for each variant. In the assays comparing the specific activity among variants in the Co(I)Cbl and Co(II)Cbl assay comparison, the enzyme concentration was 800 nM and this was calculated using the BCA protein assay kit (Pierce), as several variants lacked any tryptophans to reliably quantify their concentration by NanoDrop.</p><!><p>Purified LmEutT in buffer containing 20 mM phosphate, 200mM NaCl, and 1 mM TCEP, was mixed with Co(II)Cbl (reduced with saturated sodium formate solution) and ATP under anoxic conditions. Samples were prepared such that the final protein concentration was ~300 μM with a 0.9:1 Co(II)Cbl:Protein ratio and a 10x excess of MgATP. X-Band EPR spectra were collected on a Bruker ESP 300E spectrometer using an Oxford ESR 900 continuous-flow liquid helium cryostat and an Oxford ITC4 temperature controller. All spectra were obtained at 20 K with a modulation amplitude of 5G, modulation frequency of 100kHz, and gain of 60 dB. The EPR data were fit using Mark Nilges' SIMPOW program.37 All CD spectra were obtained at room temperature using a Jasco J-715 spectropolarimeter.</p><!><p>Structural models of LmEutT (UniProt accession code A0A0H3GFM5) and SeEutT (UniProt accession code Q9ZFV4) were constructed with Phyre2 one-to-one threading program utilizing the LrPduO structure (UniProt accession code A0A0U5JSW2, PDB code 3CI1) as the template.38 The model was subsequently downloaded and imported into MacPyMol 1.7.4.4 to generate the surface model and highlight residues of interest.</p><!><p>Amino acid homology between PduO and EutT was initially uncovered between LmEutT and LrPduO, using the NCBI pBLAST algorithm with default parameters. We note that the same program and parameters did not identify significant homology between SeEutT and LrPduO. The amino acid sequences of PduO homologues and the sequences of EutT homologues were aligned using Geneious software version 8.1.9 by employing parameters that relaxed the requirement for end-to-end sequence similarity and gap stringency (Fig. 2). This analysis revealed that all EutT homologues have an extended N-terminus compared to PduO homologues. The majority of the residues that are conserved between all EutT and PduO homologues used in the alignment correspond to LrPduO residues reported to bind the ATP nucleotide substrate and form an inter-subunit salt bridge.24</p><p>Notably, the reported ATP-binding motif in LrPduO [T5-(K/R)-X-G-D-X-G-X-(T/S)13] is not conserved across EutT homologues, with the exception of T13 in LrPduO.24 Importantly, these non-conserved residues are believed to interact with the substrate via Nα, which could explain why the side chains are not conserved. The residues whose side chains are implicated in the binding of ATP in the LrPduO structure, including T13, are conserved in the EutT and PduO homologues included in the alignment (Fig. 2, blue arrows). The only exception to this pattern involves the human adenosyltransferase (hATR), which has serine in lieu of threonine at position 13 in the LrPduO sequence.24 Additionally, the residues that form a salt bridge in the LrPduO crystal structure, i.e., D35 and R128, are also conserved in the EutT homologues, which correspond to residues D110 and R200 in LmEutT (Fig. 2, green arrows). The sequence alignment suggests that EutT ACATs may have a similar mode of ATP-binding as PduO, and may also form an inter-subunit aspartate-arginine salt bridge. This hypothesis was investigated further through site-directed mutagenesis of these residues in LmEutT followed by in vivo and in vitro analyses</p><!><p>Variants of LmEutT were constructed to change the residues presumed to be involved in ATP binding and formation of the inter-subunit salt bridge (Table S1, S2). These variants were tested for their ability to complement an S. enterica ΔACAT mutant, using a derivative of the reporter assay (Peut-lacZ transcriptional fusion) described elsewhere.8, 26 Interestingly, none of the variants synthesized enough AdoCbl to support growth under the conditions tested (Fig. 3A). To confirm that the variants were being produced in vivo, a Western blot was performed on lysates grown to mid-log phase in medium supplemented with lactose, glycerol, Cbl and ethanolamine. Although LmEutT was detected in all samples except the vector control sample, the relative detection for all variants was less than that for LmEutTWT and varied from variant to variant (Fig. 3C).</p><p>It was plausible that the reduced amounts of LmEutT variants explained the lack of growth of the S. enterica ΔACAT strain. To investigate the effects of the various residue replacements on the catalytic mechanism of LmEutT, wild-type and variant proteins were purified and tested for activity in vitro.</p><!><p>The LmEutT variants that were mutagenized at putative ATP-binding residues were purified, and kinetic parameters were obtained for ATP and HOCbl substrates using the Co(I)Cbl assay (Table 1).8</p><p>We used the reported crystal structure of LrPduO in complex with Mg(II)ATP (PDB code 3CI1) to identify contacts between the LmEutT and ATP.24 Variants LmEutTR204K and LmEutTN231L did not have detectable activity in vitro. Residue R204 of LmEutT aligned with residue R132 of LrPduO, which we posited to hydrogen bond with the ribose-ring oxygen and the α-phosphate ribose-bridging oxygen.24 Previous work showed that the LrPduOR132K variant had a ~300-fold decrease in catalytic efficiency (kcat/Km) for ATP and a ~3000-fold decrease for Cbl.16 Residue N231 of LmEutT aligned with residue N156 of LrPduO, whose carboxamide oxygen coordinates Mg(II) while its backbone nitrogen hydrogen bonds with the hydroxyl group of the γ-phosphate moiety (Figure S1).24 In LrPduO, residues R132 and N156 coordinate regions of the ATP molecule near the C-5' that must be positioned correctly for the adenosylation reaction to proceed. Residues T88 and K98 of LmEutT aligned with residues T13 and K23 of LrPduO, respectively, and in LrPduO these residues interact with the γ phosphate of ATP (Fig. S1).24 We determined increases of ~6-fold and ~22-fold in the Khalf for ATP for the LmEutTT88V
LmEutTK98Q variants, respectively (Table 1). The LmEutTE207Q variant lacked the residue that aligns with residue E135 of LrPduO, which, as shown in Fig. S1, hydrogen bonds with the amino group of adenine in ATP. Consistent with the loss of the negative charge of E207, the Khalf for ATP of LmEutTE207Q increased ~50-fold. Importantly, the turnover number (kcat) did not significantly decrease for LmEutTT88V, LmEutTK98Q or LmEutTE207Q, suggesting that substrate affinity, not the turnover rate, was affected in these variants. Noteworthy is the fact that LmEutT co-purified with ATP, and when pure protein was incubated with Co(I)Cbl without additional ATP, enough AdoCbl was produced to support growth of a ΔACAT strain.26 Several attempts to remove the ATP substrate proved unsuccessful, making it difficult to determine the binding constant of LmEutT and its variants for ATP.</p><!><p>Several changes at positions D110 and R200, which align with residues D35 and R128 of LrPduO, were purified and tested in vitro (Table 2). In LrPduO, these residues form an inter-subunit salt bridge (Fig. S2A,B).24 The Khalf for ATP of LmEutTD110N and LmEutTR200K were not significantly affected by these substitutions compared to LmEutTWT, and the kcat / Khalf decreased by no more than a log for either variant and substrate (Table 2). This finding was surprising, because the corresponding mutations in LrPduO (i.e., LrPduOD35N and LrPduOR128K) significantly perturbed the activity of the enzyme.16 For example, the LrPduOD35N variant displayed a >50-fold increase in Km and 230-fold decrease in the catalytic efficiency (kcat/Km) relative to the wild-type enzyme (Fig. S2C).16</p><p>The LmEutTD110R, R200D double-substitution variant, which was expected to retain the inter-subunit salt bridge, was inactive. This finding was consistent with the ~6 orders-of-magnitude decrease in the catalytic efficiency of the LrPduOD35R, R128D variant (Fig. S2C).16</p><!><p>We also investigated the effect of substitutions at positions D110 and R200 of LmEutT. Again, the aforementioned positions in LmEutT are equivalent to positions D35 and R128 in LrPduO (Fig. 2). To frame our findings, we note that variants LrPduOD35R and LrPduOR128D were not found to play a significant role in oligomerization of PduO, that is, substitutions at the alluded positions did not change the trimeric state of LrPduO.16 The oligomeric state of LmEutT variants was analyzed by size exclusion chromatography to determine the percentage of each oligomeric state of LmEutT variants normalized to the total amount of protein used for the analysis (Fig. 4).</p><p>LmEutTWT formed a tetramer as well as a small amount of higher molecular weight aggregate (Fig. 4).26 All LmEutTD110 and LmEutTR200 variants displayed the same oligomeric properties. However, several of the variants differed from LmEutTWT in the relative abundance of tetrameric versus aggregate form. Variants believed to preserve hydrogen-bonding interactions were not significantly affected in their aggregate to tetramer ratio. However, the LmEutTD110R and LmEutTR200D variants, for which we predicted the salt bridge would be disrupted completely, showed significantly increased aggregation (Fig, 4). The residue-swap variant, LmEutTD110R, R200D, whose activity was significantly perturbed, displayed a tetramer-to-aggregate ratio equal to LmEutTWT. Altogether, these data supported the hypothesis that the LmEutTD110 and LmEutTR200 residues interact. Additionally, these results suggested that these residues prevent LmEutT aggregation (Fig. 4).</p><!><p>In LrPduO, residue F112 (see Fig. 2) is critical for the formation of the 4C-Co(II)Cbl species, which is required to facilitate the reduction of Co(II) → Co(I) in all ACATs studied to date.29, 41, 42 Our analysis showed that an equivalent residue to LrPduO F112 was not conserved in EutT family ACATs (Fig. 2). In the absence of a divalent metal like in SeEutT, we hypothesized that LmEutT, like PduO and CobA, might also utilize hydrophobic, bulky residues to facilitate the displacement of the lower ligand.14, 42 We explored this possibility by introducing Phe → Ala and Trp → His substitutions at each phenylalanine and tryptophan residue of LmEutT. The ability of the resulting variants to confer growth was compared to that of LmEutTWT in medium supplemented with 25 nM CNCbl. Some of the LmEutT variants tested (i.e., LmEutTF39A, LmEutTF105A, LmEutTF127A, LmEutTF140A, LmEutTF183A, LmEutTF211A, LmEutTF242A) synthesized AdoCbl that supported growth of the indicator strain that was indistinguishable from that of the same strain that synthesized LmEutTWT (Fig. 5A).</p><p>Five variants (LmEutTF72A, LmEutTF97A, LmEutTF214A, LmEutTF237A, and LmEutTW238H) displayed a discernable loss of enzymatic activity as seen by the reduction in the rate of growth relative to that of the strain that synthesized LmEutTWT (Fig. 5B). To determine whether these variants were defective in displacement of the lower ligand, the variants were purified and characterized in vitro using two assays, the Co(II)Cbl assay and the Co(I)Cbl assay.26 In the Co(II)Cbl assay, the Cbl substrate is reduced from Co(III)Cbl to five-coordinate 5C-Co(II)Cbl with reduced dihydroflavins. Upon binding of the 5C-Co(II)Cbl substrate to the ACAT enzyme, the lower ligand is displaced, forming 4C-Co(II)Cbl needed for the reduction to Co(I)Cbl. In contrast, in the Co(I)Cbl assay, HOCbl is converted to Co(I)Cbl in the presence of the strong reductant Ti(III)citrate. Thus, in the Co(I)Cbl assay, the enzyme only needs to bind and position the substrates properly for the reaction to proceed. Previous work found that when CobA and PduO residues critical for lower ligand displacement were mutated to alanine, these variant enzymes were active in the Co(I)Cbl assay, but had undetectable activity in the Co(II)Cbl assay.14, 42 When LmEutTWT and the five variants identified in vivo were purified and screened using the Co(II)Cbl and Co(I)Cbl assays, three variants showed significant deviations in activity compared to LmEutTWT (Fig. 6). LmEutTF97A variant had a higher specific activity compared to LmEutTWT in both assays. This phenylalanine is adjacent to the K98 residue involved in ATP binding. Additionally, residue F97 of LmEutT is not conserved across Type II EutTs, and aligns with a conserved alanine in Type I EutTs (Fig. 2).</p><p>We did not pursue this variant further, as the relative activity of LmEutTF97A to LmEutTWT did not suggest a role for F97 in Co(II) to Co(I) reduction. As seen in figure 6, variant LmEutTF72A had similar specific activity to LmEutTWT in the Co(II)Cbl assay, but had significantly higher specific activity than LmEutTWT in the Co(I)Cbl assay (Fig. 6). While this did not immediately suggest a role in the displacement of the lower ligand, the difference in relative specific activity in the two assays for LmEutTF72A was intriguing. Additionally, the alignment shown in figure 2 indicated that F72 is located in the same region as the metal-binding site of Type I EutTs. Finally, variant LmEutTW238H had significantly diminished activity in both assays, and the activity was nearly undetectable in the Co(I)Cbl assay (Fig. 6). In the sequence alignment, this residue is conserved as a hydrophobic, bulky residue and aligns with residue F163 of LrPduO, which is located in the active site proximal to the corrin ring and is involved in corrinoid substrate binding.42 Additional variants with substitutions of residues W238 and F72 were constructed and tested in vivo and in vitro in an effort to characterize the possible involvement of these residues in 4C-Co(II)Cbl formation.</p><!><p>To analyze the contributions of residue F72 to the LmEutT activity, we constructed LmEutTF72Y and LmEutTF72H variants. The LmEutTF72Y variant was expected to maintain the relative size of the residue at position 72, while slightly increasing its polarity. Conversely, the LmEutTF72H variant was expected to significantly increase the polarity at position 72. When the medium was supplemented with 5 nM CNCbl, the strain that synthesized LmEutTF72Y displayed wild-type growth, while the strain that synthesized LmEutTF72H showed slightly reduced growth as compared to the LmEutTWT control strain (Fig. 7A). Both variants allowed for better growth outcomes than LmEutTF72A. When 25 nM CNCbl was supplemented in the medium, only the strain harboring LmEutTF72A showed a visible growth defect, as compared to the LmEutTWT control strain (Fig. 7B).</p><p>As shown in figure 6, the LmEutTW238H variant activity was very low in the Co(II)Cbl and Co(I)Cbl assays. Consistent with this information, LmEutTW238H failed to synthesize enough AdoCbl to support growth (Fig. 5, Fig. 7). If we assume that in LmEutT the W238 residue is positioned under the cobalt, then substituting the indole side chain of tryptophan for the imidazole side chain of histidine could allow for the formation of a coordination bond between a nitrogen of the imidazolyl moiety of histidine and the cobalt ion of Co(II)Cbl, as it occurs with "base off/His on" AdoCbl-dependent isomerases.43 To determine whether this was the case, the tryptophan residue was replaced with an alanine or a leucine residue. Neither LmEutTW238A nor LmEutTW238L allowed growth in the presence of 5 nM or 25 nM exogenous CNCbl (Fig. 7), suggesting that the residue W238 was critical for activity, as these substitutions to other nonpolar residues did not improve activity in vivo. Shown in figure 8, are the results of Co(I) and Co(II) activity assays performed with LmEutTF72 and LmEutTW238 variants.</p><p>Substitution of the bulky phenyl group at position 72 by an alanine (F72A) resulted in an enzyme that had comparable activity to LmEutTWT in the Co(II)Cbl assay, but had >2-fold increased specific activity relative to LmEutTWT in the Co(I)Cbl assay (Fig. 8A). In contrast, replacement of F72 with other bulky residues (e.g., Tyr or His) resulted in variants with slightly lower specific activity in both assays, relative to LmEutTWT. The reaction rate of ACATs belonging to the CobA and PduO families are limited by the rate of corrinoid reduction. Once the corrinoid is reduced, the nucleophilic attack occurs quickly, and this is reflected in the increase in specific activity in the Co(I) assay compared to the Co(II) assay. Assuming that this is the case for LmEutT, these results suggested that residue F72 might restrict access to the active site, since replacement with a less bulky residue (e.g., Ala) increased the specific activity of the variant only in the fast step of the reaction, i.e., the nucleophilic attack (compare F72A in Fig. 8A
vs
8B).</p><p>LmEutTW238A and LmEutTW238L variants showed reduced specific activity in both assays, as compared to LmEutTWT. However, the activity of LmEutTW238H was reduced to a greater extent in both assays (Fig. 8). The alignment of this region with LrPduO suggested that residue W238 might enhance the affinity of the enzyme for the corrinoid substrate.42</p><!><p>Previous studies revealed that LmEutTWT complexed with ATP converts Co(II)Cbl to a four-coordinate species, so as to facilitate the thermodynamically-challenging Co(II) → Co(I) reduction.28 As the single unpaired electron in Co(II)Cbl resides in the Co-dz2-based molecular orbital that is oriented along the α-ligand–Co bond axis, X-band electron paramagnetic (EPR) spectroscopy provides a uniquely sensitive probe of perturbations to the axial ligation of Co(II)Cbl. Therefore, EPR studies were conducted to assess if the F72X and W238H LmEutT variants were able to displace the α-ligand in 5C-Co(II)Cbl to form 4C-Co(II)Cbl.</p><p>Consistent with the observation that the replacement of F72 with other residues decreased the specific activity in the in vitro Co(II)Cbl assay, our EPR spectra (Fig. 9, Table 3) show that the F72A, F72Y, and F72H substitutions reduce the ability of LmEutT to convert 5C-Co(II)Cbl to 4C-Co(II)Cbl relative to the WT enzyme (which achieves a >98% 5C→4C conversion.)28</p><p>The EPR spectrum of Co(II)Cbl in the presence of LmEutTF72A (Fig. 9C) shows that the F72A substitution results in a mixture of base-off 5C-Co(II)Cbl and 4C-Co(II)Cbl in the enzyme active site. While the g values for the 5C fraction are more reminiscent of base-on 5C-Co(II)Cbl (Fig. 9A) than its base-off analogue (Figure 9B), the lack of 14N (I = 1) superhyperfine splittings in the 3000–4000 G region indicates that a solvent-derived water molecule rather than the intramolecular DMB base serves as the α-ligand in this species. The unexpectedly small g spread displayed by this species likely reflects a compressed Co–O(H2) bond relative to free base-off 5C-Co(II)Cbl, as this would destabilize the Co-dz2-based molecular orbital and thus decrease the extent of spin-orbit mixing between the ground and ligand field excited states.44 The fraction of 4C-Co(II)Cbl bound to LmEutTF72A gives rise to a series of weak features across the entire 1000–4500 G range, as reported previously for LmEutTWT and other ACATs.28, 29, 41, 45 This 4C-Co(II)Cbl signal is more easily identified in the EPR spectrum of Co(II)Cbl bound to the F72Y variant (Fig. 9D), as the F72Y substitution only marginally compromises the ability of LmEutT to convert Co(II)Cbl to a 4c species (~80% 5C→4C conversion, Table 3). Notably, the fraction of 5C-Co(II)Cbl that is formed in this variant is characterized by g values that are similar to those of free base-off 5C-Co(II)Cbl, indicating a relatively unperturbed Co–O(H2) bond. Finally, the EPR spectrum of Co(II)Cbl in the presence of LmEutTF72H (Fig. 9E) reveals contributions from 4C-Co(II)Cbl (~45%) and two distinct base-off 5C-Co(II)Cbl species, one with a normal Co–O(H2) bond (~35%) and one in which this bond is compressed (~20%). The fact that a substantial fraction of 4C-Co(II)Cbl is generated in each of these variants supports the hypothesis that F72 restricts access to the active site and is not essential for the conversion of 5C-Co(II)Cbl to 4C-Co(II)Cbl.</p><p>EPR spectroscopy was also used to probe the origin of the low activity of LmEutTW238H displayed in both assays. One cause of low activity could be the replacement of the intramolecular base with His-238 similar to what has been observed for enzymes binding the Cbl cofactor in the base off, His-on form such as MCM. However, the EPR spectrum of Co(II)Cbl in the presence of LmEutTW238H (Fig. 9F) exclusively has contributions from a base-off 5C-Co(II)Cbl species, indicating that the drastically reduced activity in the Co(II)Cbl assay might arise from an inability of the W238H variant to form appreciable amounts of 4C-Co(II)Cbl. To probe whether the inability of LmEutTW238H to convert 5C-Co(II)Cbl to 4C-Co(II)Cbl was temperature dependent, we also collected room temperature circular dichroism (CD) data. Surprisingly, the room temperature CD spectra of Co(II)Cbl in the absence and presence of LmEutTW238H are nearly identical (Fig. S3), suggesting that the significantly decreased specific activity of this variant in both the Co(II)Cbl and Co(I)Cbl assays is due to weak binding of the Cbl substrate to the enzyme active site.</p><!><p>The amino acid sequences of LmEutTWT and SeEutTWT were modeled to the LrPduO structure using Phyre2 (Fig. 10).38 The software did not align the N-terminal residues of LmEutT or SeEutT since they were not present in LrPduO, and these were excluded from the model. However, the models suggest that there are structural similarities between PduO and EutT ACATs, particularly in the substrate-binding region (Fig. 10).</p><!><p>The sequence alignment of EutT and PduO ACATs and the subsequent biochemical analysis of variants with substitutions at the conserved ATP-binding residues suggest that EutT and PduO ACATs evolved similar substrate-binding sites. The similarities were not noted before as the identified ATP-binding motif in LrPduO is not conserved in EutT family ACATs. However, the five residues that were noted in LrPduO to bind to the ATP substrate are conserved across the PduO and EutT homologues included in the alignment. Of note is the conserved NRLS(D/S) motif near the C-terminus of both ACAT families; this motif is part of one of the core α-helices in the structure in LrPduO and may suggest that these ACATs are evolutionarily related.</p><!><p>The stability of the LrPduO oligomer is not dependent on the presence of the salt brige between residues D35 and R128. In contrast, we found that the stability of the LmEutT oligomer is affected by substitutions of these residues. The LmEutTD110R and LmEutTR200D variants formed significantly more aggregate than variants LmEutTD110N, LmEutTR200K, or LmEutTWT (Fig. 4). If formation of the salt bridge was preserved, as in the LmEutTD110R,R200D variant, the oligomeric state mimicked that of LmEutTWT. Contrary to LrPduO, substitutions of residues D35 or R128 for LmEutT affected the Km of Co(II)Cbl more than the Km of ATP (Table 2). These observations suggest that the salt bridge may play a greater role in LmEutT in the stability of the oligomer and affinity for Cbl than in LrPduO.</p><!><p>The mechanism of base displacement has been well documented in CoA and PduO type ACATs, where a phenylalanine and/or tryptophan residue is involved.14, 42 These residues disrupt the (alpha) ligand coordination which allows the reduction of Co(II) to Co(I) to proceed. Analysis of all the tryptophan and phenylalanine residues in LmEutT did not identify a residue that possessed similar biochemical characteristics to previously studied variants of F112 in LrPduO (Fig.7–8).42 Based on these observations and the spectroscopic differences between EutT ACATs and the other ACAT families, we suggest that Class II EutT ACATs utilize an alternative mechanism for base displacement. Our phenotypic and biochemical observations suggest that F72 may be located near the entrance of the substrate-binding site and may control Cbl access to the active site, while residue W238 may be located within the substrate-binding pocket and hence contribute to the hydrophobic pocket for corrin ring positioning.</p><p>The unique mechanism of ligand displacement by LmEutT as revealed in the present study is consistent with results obtained previously for SeEutT and LmEutTWT.28, 29 Specifically, it was found that these enzymes can bind both Co(II)Cbl and Co(II)Cbi+; however, only Co(II)Cbl is converted to a 4C species and can thus be adenosylated in vivo and in the in vitro Co(II)Cbl assay. On the basis of this observation, it was concluded that members of the EutT family of ACATs possess a binding pocket for the DMB base to enhance their affinity for the native substrate and promote axial ligand dissociation. The EPR data obtained in the present study provide evidence that in LmEutT, residues F72 and, in particular, W238 are required for maximum 5C → 4C-Co(II)Cbl conversion. Although in the absence of a crystal structure the location of these residues relative to the Cbl substrate cannot be determined, our results for the LmEutTW238H variant are consistent with W238 playing an important role in properly organizing the putative DMB binding pocket.</p><!><p>This work provides insights into how the Class II EutT ACAT from Listeria monocytogenes (LmEutT) binds its substrates. The residues that bind ATP and form an inter-subunit salt bridge in LrPduO are conserved in LmEutT, and in vitro studies of LmEutT variants suggest that these residues perform the same roles in LmEutT as they do in LrPduO. Interestingly, mutations to the inter-subunit salt bridge residues D110 and R200 did not affect LmEutT in the same manner as in LrPduO. Differences in oligomeric state (LrPduO forms a trimer and LmEutT a tetramer) may contribute to these differences. Lastly, residues F72 and W238 were identified as needed for Cbl binding, however, unlike in PduO and CobA, LmEutT does not use a canonical residue to generate 4C-Co(I)Cbl. The mechanism of 4C-Co(I)Cbl formation remains unclear.</p>