Thiolation-enhanced substrate recognition by D-alanyl carrier protein ligase DltA from Bacillus cereus

Cloning, protein preparation and crystallization

All reagents were from VWR unless specified otherwise. The wild-type as well as the C269A mutant of DltA from B. cereus was cloned for over-expression of BcDltA as described previously¹⁷. The pET28-BcDltA construct carries an Ala-1 mutation at the N-terminus and eight extra residues at the C-terminus (LEHHHHHH). The soluble fraction of BcDltA was purified by nickel-affinity chromatography followed by gel filtration. BcDltA was concentrated to ~20 mg/mL by ultra-filtration.

Crystallization and structure determination

The concentrated BcDltA protein was crystallized using the hanging drop crystallization method at a room temperature of 21°C. The optimal well solution for crystallization contained 0.1 M MgCl₂, 0.5 M KCl, 16% polyethylene glycol (PEG) 3,350 (Sigma-Aldrich) and 0.05 M Hepes-NaOH buffer at pH 7.2. Each drop was composed of 1 μL of protein and 1 μL of well solution. The plate-shaped crystals grew to a maximal size of 0.4 mm × 0.3 mm × 0.05 mm in three days. Crystals were gradually transferred to stabilizing solutions composed of the crystallization well solution supplemented with 8%, 16% and 24% glycerol, soaked for 1 minute, then flash-cooled to - 173.15°C in a nitrogen stream generated by an Oxford CryoSystems device. A total of 400 0.4-degree oscillation images were acquired and processed using a Brukers Proteum-R system as already described²⁷. The previously solved BcDltA model (PDB code 3DHV)¹⁷ was used as the starting model for two-domain rigid body refinement followed by positional refinement using Crystallography & NMR System (CNS)²⁸. This resulting model was subjected to ten cycles of rebuilding and refinement using Arp/Warp²⁹. The rebuilt model was iteratively rebuilt using XtalView³⁰ and then refined using CNS²⁸. The final model had 90.8% of the residues in the most favored regions on a Ramachandran plot. Val-301 with clear electron density and Asp-336 with blurry electron density were the only two residues found in the disfavored region. Statistics of the diffraction data, refinement and geometry are listed in Table 1. The molecular figures were generated using Molscript³¹ and rendered using Raster3D³². The coordinates and structure factors have been deposited in the Protein Data Bank^33–35 (entry code 4PZP).

Tryptophan fluorescence measurement

The intrinsic tryptophan fluorescence of 1.0 ml 0.4 uM BcDltA solution with 0 to 2 mM ATP was acquired at a room temperature of 21°C using a PerkinElmer LS-55 fluorescence spectrometer. The excitation wavelength was 305 nm and a fluorescent emission in the 310 nm to 390 nm range was recorded. The relative fluorescence increase at 345 nm was used to quantify the ATP-bound fraction of BcDltA. Assuming that the fluorescence gain is proportional to [ATP]/(K_D + [ATP]), Prism software (GraphPad Software) was used to derive the dissociation constant of ATP.

Pyrophosphate quantification assay

As previously described, pyrophosphate released from the adenylation reaction is broken down into phosphate by pyrophosphatase¹⁷. The resulting phosphate was quantified by a dye solution containing 0.033% w/v Malachite Green, 1.3% w/v ammonium molybdate and 1.0 M HCl³⁶. The 200 μL reaction solutions contained 5 μM BcDltA, 0.1 M KCl, 0.01 M MgCl₂, 0.05 M Tris-Hepes buffer at pH 7.2, 5 unit/mL of inorganic pyrophosphatase from baker’s yeast (Sigma-Aldrich) and specified concentrations of D-alanine, ATP and CoA (Sigma-Aldrich). A volume of 25 μL of reaction solution was retrieved every 3 or 5 minutes and mixed thoroughly with 475 μL of the dye solution. The absorption at a wavelength of 620 nm was recorded after 90 seconds. The initial rates (1/2 of the phosphate concentration increase per minute) of the adenylation reaction were derived from the time courses of phosphate accumulation. The correlation between initial reaction rate and substrate concentration was fitted with Michaelis-Menten equation using the Prism software (GraphPad Software).

Thiol quantification assay

The free thiol group of CoA was quantified as described previously²⁴ by a dye solution composed of 1 mM 5,5’-dithio-bis (2-nitrobenzoic acid) (DTBN), or Ellman’s reagent (Sigma-Aldrich) and 50 mM Tris-EDTA solution at pH 8.0. Absorption at a wavelength of 412 nm was used to quantify the concentration of free thiol group. The reaction rate was derived by the rate of thiol depletion. The correlation between initial reaction rate and substrate concentration was also fitted with Michaelis-Menten equation using the Prism software.

Crystal structure of substrate-free BcDltA

The same DltA protein from B. cereus with a C-terminal hexahistidyl fusion tag used in our previous crystallographic studies on BcDltA^17,24 was crystallized in the absence of ATP, D-alanine or CoA. One crystal diffracted to 1.9 Å resolution and belonged to space group P2₁ (Table 1), the same space group as in previously reported crystals of BcDltA in complex with D-alanine adenylate¹⁷ and with ATP²⁴. Despite having 8 Å shorter crystallographic a axis, 5 Å shorter b axis and 5° smaller β angle than the previously reported crystal of DltA/D-alanine adenylate complex, the structure was successfully solved by rigid-body refinement using the previously determined BcDltA structure¹⁷ (PDB code 3DHV) as the starting model. The 504-residue BcDltA structure can be divided into two domains (Figure 2): an N-terminal major domain from the N-terminus to Asp-399, and a C-terminal minor domain from residue 400 to the C-terminus. The disposition of the two domains in the substrate-free BcDltA structure remains similar to that of the starting model (Figure 2A). The electron density map indicated several disordered regions (Ser-153 to Pro-159, Pro-363 to Glu-367, Arg-397 to Glu-413, Lys-433 to Tyr-440) with the corresponding regions in the starting model highlighted in magenta in Figure 2B. The first disordered region is part of a highly conserved P-loop (Thr-152 to Lys-160) found in homologous AMP-forming proteins^37–39. Due to its similar amino acid composition (glycine, serine, threonine and lysine) to that of P-loop or Walker A motif found in ATPases and GTPases⁴⁰, this loop has long been thought to catalyze the adenylation reaction. In the crystal structure of human medium-chain acyl-CoA synthetase in complex with ATP, this loop intimately interacts with the β- and γ-phosphates of the ATP substrate⁴¹. Functional relevance of regions 363–367 and 433–440 are unknown. The former is located at a 2-turn helix at the surface of the N-terminal domain (Figure 2B). The latter is interacting with the longest disordered inter-domain region in this structure (397–413) which contains several key elements of DltA. Arg-397 has been observed to interact with the β-phosphate of ATP and to play an important role in catalysis²⁴. Asp-399, equivalent to Asp-398 in BsDltA, serves as the hinge residue for domain rotation. As observed for the equivalent Asp-402 of 4-Chlorobenzoate-CoA ligase¹⁵, the rotation around main-chain single bonds in this hinge residue could account for a 146° swing of the C-terminal domain as we compared the crystallized adenylation conformation of BcDltA and thiolation domain of BsDltA (Figure 1, bottom with the disordered inter-domain linker in green). The equivalent main-chain atoms in the N-terminal domains of BcDltA in adenylation conformation (PDB entry 3DHV) and BsDltA in thiolation conformation (PDB entry 3E7W) are superposed with a root-mean-deviation of 0.97 Å, and the deviation for the C-terminal domain was 1.00 Å. These values indicate that there is no dramatic conformational change within each domain in addition to the 146° rotation around the hinge aspartate residue. The C-terminal part of this flexible inter-domain region also contains a β-hairpin which has been observed to interact with CoA in homologous acetyl-CoA synthetase²³.

Figure 2. Structure of DltA in the absence of substrate.

The ribbons representation of previously reported BcDltA structure (PDB entry: 3DHV) is shown with D-alanine-adenylate and surrounding side-chains in ball-and-stick model. The N-terminal domain is shown in gray, and the C-terminal domain in gold. A. The substrate-free structure of BcDltA is superimposed on the BcDltA/D-Ala-AMP complex. The N- and C-terminal domains are colored in cyan and magenta, respectively. B. The four corresponding regions of the BcDltA/D-Ala-AMP complex, which are disordered in the substrate-free form of BcDltA, are highlighted in magenta.

K_M and k_cat of BcDltA in the presence and absence of D-alanyl carrier CoA

In our previous study, we have verified that CoA can mimic D-alanyl carrier protein DltC²⁴, as also discovered for DltA homologs⁴². In that study, we have observed that the reaction rate is increased by nearly an order of magnitude by the presence of saturating concentration of CoA, which is explained by the faster release of the thiolation product rather than by release of the adenylation intermediate. In order to get a comprehensive understanding of the effects by CoA as the DltC mimic, we further studied the enzymatic properties of BcDltA in the presence of a saturating concentration (5 mM) of ATP or D-alanine, and in the absence of CoA or in the presence of a saturating concentration (5 mM) of CoA. The reaction rates derived from the pyrophosphate accumulation assay and the thiol depletion assay were similar (Figure 4). The thiolation assay was noticeably noisier than the pyrophosphate assay and we therefore limit the discussion to K_M and k_cat values derived from the pyrophosphate assay. Somewhat unexpectedly, BcDltA showed much higher apparent affinity, or decreased K_M value, towards ATP (0.46 mM to 0.01 mM) and D-alanine (1.1 mM to 0.03 mM) in the presence of 5 mM CoA (Figure 3 and Figure 4, Table 2). On the contrary, the apparent affinity towards L-alanine decreased in the presence of CoA, with K_M increased from 14.4 mM to 109 mM (Figure 4 and Table 2).

Figure 3. Rate of pyrophosphate release and ATP-induced fluorescence gain.

The reaction solutions contained 0.005 mM wild-type BcDltA, specified concentrations of ATP and CoA, 5 mM D-alanine, 0.1 M KCl, 0.01 M MgCl₂, 0.05 M Hepes-NaOH buffer at pH 7.2, 5 unit/mL of yeast inorganic pyrophosphatase. A. The initial rates of pyrophosphate accumulation divided by the BcDltA concentration are shown. The reaction rates in the presence of CoA are taken from a previous study²⁴. B. Relative fluorescence gains as a fraction of the fluorescence intensity in the absence of ATP are shown.

Figure 4. Rate of pyrophosphate release and thiol depletion.

The reaction solutions contained 0.005 mM wild-type BcDltA, 5 mM D-alanine, 0.1 M KCl, 0.01 M MgCl₂, 0.05 M Hepes-NaOH buffer at pH 7.2, 5 unit/mL of yeast inorganic pyrophosphatase and specified concentrations of ATP, alanine and CoA. Reaction rates for wild-type BcDltA are shown on the left, and those for the C269A mutant protein are shown on the right. The reaction rates in the absence of CoA are taken from a previous study¹⁷.

Relaxed D-alanine preference by the C269A BcDltA mutant protein

The side-chain of Cys-269 sits at the bottom of the D-alanine-binding pocket which may make VDW clash with the methyl side-chain of L-Alanine^16,17. We also studied the effect of CoA on D- and L-alanine preference of the C269A mutant of BcDltA (Figure 4 and Table 2). In the presence and absence of CoA, the C269A protein showed relaxed preference for D-alanine over L-alanine. As observed for the wild-type protein, CoA also enhances the D-alanine preference of this BcDltA mutant protein. However, the CoA-induced changes in Michaelis constant K_M were less dramatic (3.1 mM to 0.50 mM for D-alanine, 6.6 mM to 8.8 mM for L-alanine) than those observed for the wild-type protein.

ATP binding by BcDltA

Change in tryptophan fluorescence of BcDltA was minimal in the presence of ATP in the 10 micromolar range. We then found that at an excitation wavelength of 305 nm, the absorption of up to 2 mM of ATP was negligible and there was fluorescence gain associated with increasing concentration of ATP. The fluorescence gain-derived dissociation constant K_D for ATP (0.43 mM) was similar to the Michaelis constant K_M (0.46 mM) derived in the absence of CoA (Figure 3). There was no detectable fluorescence change to derive K_D values for D-alanine. For CoA, we were not able to isolate fluorescence change from absorption by CoA in millimolar concentration.

DltA strongly prefers D- over L-alanine

Bacteria selectively incorporate D- over L-alanine in cell wall components. There appears to be no exception in ubiquitous esterification of lipoteichoic acids by alanine²⁵. The enantiomer selectivity of BcDltA observed in the absence of any D-alanyl carrier¹⁷, however, has been intriguingly mediocre. The newly acquired kinetic data in the presence of saturating CoA, a DltC mimic, shows that the k_cat values are less than 2-fold different for D- and L-alanine (10.9 and 19.3 min^-1) while the K_M values are more than 1000-fold different favouring D-alanine over L-alanine (0.03 and 109 mM). Adding the fact that the intracellular concentration of D-alanine (in the order of 10² μM) is approximately 10-fold more abundant than the L-enantiomer (in the order of 10¹ μM)⁴³, DltA functioning at a saturating concentration of the D-alanyl carrier protein DltC would favor the ligation of the D-enantiomer by approximately 4 orders of magnitude. Such striking enantiomer selectivity is consistent with the much lower L-alanine content found in lipoteichoic acid²⁵. A recent study has shown that the dlt operon is induced by cell envelope stress such as acidic pH and antibiotics⁴⁴. It is possible that stress-induced expression of DltC may reach a saturating concentration for interacting with DltA and therefore ensure the almost exclusive enantiomer selectivity as observed in the presence of the DltC mimic. The comparison between the kinetic properties of the wild-type and the C269A mutant proteins also supports the notion that Cys-269, and its equivalence in other DltA proteins, contributes to the enantiomer selectivity of DltA^16,17.

Thiolation conformation of DltA is compatible with adenylation substrates

It is satisfying to observe the stringent D-alanine preference. At the same time, it is also puzzling to appreciate that such selectivity on the chirality of alanine, a substrate of the adenylation reaction, can only be achieved in the presence of CoA, a substrate of the thiolation reaction. The intrinsic fluorescence of tryptophan in BcDltA enabled us to derive the dissociation constant K_D for ATP in the absence of other substrates. The new form of crystal structure in the absence of any substrate is noticeably more disordered than the previously observed adenylation conformation of BcDltA¹⁷ and thiolation conformation of BsDltA¹⁶. The longest such disordered region is between Arg-397 and Glu-413, which contains the inter-domain hinge residue Asp-399, interacts with β-phosphate of ATP, and forms the pantetheine channel. Possibly, this important region remains disordered in the presence of saturating D-alanine, therefore providing an explanation for the similar values between the above mentioned K_D (0.43 mM) and the K_M (0.46 mM) for ATP, in the presence of saturating D-alanine but in absence of CoA. The more disordered nature of the substrate-free conformation of BcDltA also implies that the previously observed adenylation and thiolation conformations are intrinsically unstable unless stabilized by the interaction with one or more substrates. This structural feature of BcDltA likely explains the relatively low sub-millimolar affinity for ATP, since part of the stabilizing BcDltA-ATP interactions would be used to compensate the cost of establishing the adenylation conformation of the protein.

BcDltA is a very slow enzyme. Unless the substrate dissociation step happens to be extremely slow as well, we could approximate the observed K_M values to the K_D values of corresponding BcDltA-substrate intermediates, and therefore enable reasoning in the context of structural stability of such intermediates. As such, we reasoned that the approximately one order of magnitude difference in K_M values for the adenylation substrates ATP and D-alanine observed in the presence and absence of the thiolation substrate may imply the existence of a quadruple intermediate of the DltA enzyme in complex with all three substrates, which may be markedly different from a ternary intermediate of BcDltA with the two adenylation substrates.

We then resorted to three-dimensional model building so as to answer the question on which of the adenylation and thiolation conformations may be compatible with binding to all three substrates. The BcDltA/D-alanine-adenylate complex (PDB entry 3DHV)¹⁷ was chosen as the adenylation conformation and as the reference set of atomic coordinates. The adenylated intermediate was dissected to generate the D-alanine model. The N-terminal domain of the BcDltA/ATP complex (PDB entry 3FCE)²⁴, which is also in the adenylation conformation, was superposed on the reference set to orient the ATP substrate. The N-terminal domain of the BsDltA/AMP complex (PDB entry 3E7W) was also superposed on the reference set to derive the re-oriented thiolation conformation. Main-chain atoms equivalent to those interacting with AMP in BcDltA (270–272, 292–299) in the quadruple complex of acetyl-CoA synthetase in its thiolation conformation (PDB entry 2P2F)²³ were superimposed on the reference set to orient the CoA model. In the adenylation conformation (Figure 5A, the pantetheine channel is apparently blocked by the main-chain atoms immediately preceding the catalytic Lys-492 of BcDltA (Lys-491 of BsDltA). Although we could not completely rule out the possibility that an allosteric site for CoA exists, no such site has ever been observed for this superfamily of enzymes. Therefore, a quadruple complex in the adenylation conformation is unlikely. The thiolation conformation (Figure 5B), on the other hand, appears to be compatible with binding ATP so long as Arg-408 of BcDltA (Arg-407 of BsDltA) adopts another rotamer. It has been previously observed that the homologous acetyl-CoA synthetase in its thiolation conformation binds AMP, acetate and CoA²³, and that BsDltA in its thiolation conformation binds AMP and appears to have a well-formed D-alanine-binding pocket¹⁶. These structural evidences seem to suggest that a quadruple intermediate may form with the enzyme in its thiolation conformation but not in the adenylation conformation. Since the thiolation conformation has an AMP-binding site and its N-terminal domain, which provides most of the ATP-interacting residues such as the P-loop and Arg-397, remains essentially identical to that that in the adenylation conformation, it is not surprising that only one arginine side-chain is required to adopt another rotamer to accommodate ATP. This arginine residue (Arg-408 of BcDltA) forms a salt bridge with the divalent cation-anchoring side-chain of Glu-298 in BcDltA (Glu-297 in BsDltA)²⁴, which appears to be an important structural feature to modulate the conformational change¹⁶. Another arginine residue (Arg-397 of BcDltA, Arg-396 of BsDltA) also seems to facilitate the conformational change. It forms a salt bridge with the hinge aspartate residue in the thiolation conformation. In the adenylation conformation, this arginine side-chain adopts a more extended rotamer and forms a salt bridge with the β-phosphate group of ATP (Figure 5A). ATP binding would result in re-orientation of both arginine residues and disruption of two salt bridges, thus mobilizing the thiolation conformation.

Figure 5. Structural model of DltA in complex with ATP, D-alanine and CoA.

Cα traces of the two modelled quadruple complexes are shown in stereo. Except for the green-colored region between Arg-397 and Glu-413, the N- and C-terminal domains are colored in gray and salmon, respectively. The three substrates and selected side-chains are shown in ball-and-stick model. A. The quadruple complex in adenylation conformation. Residue numbers in BcDltA are shown. B. The quadruple complex in thiolation conformation. Residue numbers in BsDltA are shown.

Structural basis for CoA-enhanced affinity for ATP

The majority of ATP-binding elements lie in the N-terminal domain which remains similar in both adenylation and thiolation conformations. The most significant structural feature in the C-terminal domain for binding ATP is the catalytic Lys-492 of BcDltA (Lys-491 of BsDltA) in the adenylation conformation. In the thiolation conformation, the catalytic residue is replaced by Lys-403 (Lys-402 of BsDltA) (Figure 5). In addition, the thiolation conformation is more ordered than the substrate-free conformation. The favourable DltA/ATP interactions may no longer be used to compensate the energetic cost of stabilizing the disordered hinge region. Therefore the affinity for ATP by the CoA-bound BcDltA in its thiolation conformation should be higher than substrate-free BcDltA.

Structural basis for CoA-enhanced enantiomer selectivity for D-alanine

For both the wild-type and C269A mutant BcDltA, the presence of saturating CoA increases the apparent affinity for D-alanine while decreasing affinity for L-alanine. The majority of alanine-interacting residues lie in the N-terminal domain. The amino group of D-alanine is stabilized by Asp-197 of BcDltA (Asp-196 of BsDltA). Cys-269 of BcDltA (Cys-268 of BsDltA) lies close to Cα of D-alanine and serves as one major determinant of enantiomer selectivity. The carboxylate group of D-alanine is stabilized by Lys-492 in the adenylation conformation or by Lys-403 in the thiolation conformation. At the end of the pantetheine channel, Phe-196 of BcDltA (Phe-195 of BsDltA) adopts different rotamers in the two conformations. In the adenylation conformation of BcDltA/D-alanine adenylate, the shortest distance from Phe-196 side-chain to methyl group of D-alanine is 4.15 Å. In the modelled quadruple complex in the thiolation conformation, the Phe-196 side-chain does not contact D-alanine but the thiol group of CoA lies in closer proximity of D-alanine side-chain (3.34 Å), as expected from the pending thiolation reaction between the thiol group of CoA and the carbonyl group of D-alanine-adenylate. On the other hand, the tighter alanine-binding pocket may exert stronger VDW repulsion toward L-alanine, thus lowering further the affinity for the wrong enantiomer. Interestingly, Cys-269 also has a thiol group. The removal of this group in the C269A mutant of BcDltA reduces the affinity for D-alanine and increases the affinity for L-alanine by approximately one order of magnitude, which is almost exactly the opposite to the effect of introducing the thiol group of CoA in the alanine-binding pocket. Since sulphur atoms are larger and more inducible than the second-period elements carbon and oxygen, it is not surprising that the VDW interaction involving a thiol group makes a significant impact on the stability of enzyme/substrate complex.

Hypothesized enzymatic cycle of DltA

Intracellular concentration of D-alanine⁴³ generally exceeds the K_M value in the presence of saturating ATP and CoA. Typical intracellular concentration of ATP lies in the millimolar range⁴⁵, exceeding the K_M value for ATP as well. The concentration of the possibly stress-induced DltC⁴⁴ may also reach saturating level in bacteria when such stress is present. As implied by the CoA-triggered dramatic change in K_M values for ATP and D-alanine, the two adenylation substrates are likely incorporated by the enzyme with pre-bound CoA rather than with merely the other adenylation substrate. In addition, three-dimensional modelling suggests the CoA-bound state of the enzyme can only exist in its thiolation conformation. We therefore hypothesize a two-conformation model for the enzymatic cycle catalyzed by DltA (Figure 6) in the presence of saturating concentration of the three substrates. Our model differs from the three-conformation model proposed for DltA and adenylation domains in non-ribosomal peptide synthetase¹⁶ which includes a third substrate-free conformation. This model also differs from the two-conformation model proposed for and for 4-chlorobenzoate-CoA ligase^26,46 which includes substrate-free state of the enzyme. The two previously proposed models are consistent with a typical Ping-Pong mechanism. Neither a substrate-free conformation nor a substrate-free state of the enzyme is required in our model for the enzymatic cycle of DltA once the protein enters the reaction cycle. The adenylation reaction starts from a ternary complex with ATP and D-alanine and proceeds with the release of pyrophosphate. A domain rotation around the aspartate hinge residue follows transforming into the thiolation conformation which binds CoA, or DltC in bacteria, and catalyzes the thiolation reaction with D-alanine by displacing AMP. In our model, the resulting complex with the two thiolation products proceeds with AMP release, D-alanine-CoA/CoA exchange, D-alanine binding, and ATP binding. In the quadruple complex, the ATP-bound magnesium ion would replace Arg-408 of BcDltA in forming a bridge with Glu-298, as observed in the crystal structure BcDltA/ATP complex²⁴. The disruption of the Arg-408 to Glu-298 salt bridge would destabilize the thiolation conformation and facilitate a reverse domain rotation and release of CoA which is not compatible with the adenylation conformation. The hypothesized D-alanine-CoA/CoA exchange step is the central piece of this enzymatic cycle, which reflects the finding that CoA affects DltA’s apparent affinities with D-alanine and ATP. If DltA were to become substrate-free for a long enough period of time, it would form a ternary complex with ATP and D-alanine, which contradicts the observed effect by the thiolation substrate CoA. It is worth noting that the sequence of D-alanine and ATP binding is uncertain. Similarly, AMP release has to occur before ATP binding but not necessarily before exchange with CoA or D-alanine binding.

Figure 6. Hypothetical enzymatic cycle of DltA.

Non-covalent DltA complexes are shown in text. Forward reaction, substrate binding and release steps are shown by arrows. Steps fulfilled by the adenylation conformation are shown in the green box, and those by the thiolation conformation in brown. The thioester between D-Ala and CoA (D-Ala-CoA) is the final product.

The hypothesized enzymatic cycle involves a second CoA binding step, and a CoA release step in addition to the Ping-Pong mechanism previously proposed for DltA and its homologs. Both additional steps seem unnecessary for the enzymatic reaction itself, but are required to explain our enzymatic data and are consistent with the three-dimensional models of BcDltA and BsDltA. A Ping-Pong mechanism would require that the binding of CoA and the binding of either adenylation substrate be uncompetitive, and therefore the apparent K_M and k_cat values for either adenylation substrate both would become larger at higher CoA concentration. While the k_cat values for both ATP and D-alanine did become larger at saturating CoA concentration, the K_M values actually became smaller, therefore contradicting the typical Ping-Pong mechanism. Another property of aminoacyl-CoA synthetases including DltA is the inhibitory effect of CoA at high concentration⁴², which is difficult to explain by a typical Ping-Pong mechanism unless the enzymatic cycle includes an additional CoA-dissociation step as in our model. The closest homologs of DltA are amino acid-activation domains found in non-ribosomal peptide synthetases⁴². Similar to DltA, these homologs also pass the adenylated intermediate to the 4’-phosphopantetheine group attached to a serine residue on a peptide carrier domain. It is possible that such amino acid activation domains may also act as DltA.

The extra binding step for 4’-phosphopantetheine D-alanyl carrier could serve as the sensor for the availability of the carrier. The maximum rate catalyzed by BcDltA is approximately seven times faster in the presence of CoA than in its absence, with respective k_cat values 10.9 min^-1 and 1.5 min^-1. Moreover, the intracellular concentration of D-alanine is typically found in the 100 micromolar range⁴³, which lies above the K_M value for D-alanine in the presence of CoA (~30 µM) but below the K_M value in the absence of CoA (1100 µM) (Table 2). The reaction rate should become slower by approximately 100 fold when the thiol carrier is absent. It is worth noting that the adenylated D-alanine intermediate generated by the adenylation reaction is not covalently attached to the enzyme and could be released and wasted when the thiolation substrate is absent. The significant slowing down of the adenylation reaction in the absence of the 4’-phosphopantetheine carrier therefore provides a biological advantage.