大家好，今天为大家带来的是2024年4月11日发表在“Green Chemistry”上的“Mild hydrolysis of chemically stable valerolactams by a biocatalytic ATP-dependent system fueled by metaphosphate”，通讯作者是奥地利大学的Mélanie Hall。
Compared to the smaller β-lactam, medium- and 6-membered cyclolactams are molecules with significant stability. As monomers, they allow the use of nylon-4 and nylon-5, which are widely used alternative polyamides to caprolactam-nylon-6. The chemical hydrolysis of monocyclic γ-lactam and δ-lactam into the corresponding amino acids requires harsh reaction conditions, and so far, no mild (enzymatic) protocols have been reported. Herein, the biocatalytic potential of a pair of heterologous expressed bacterial ATP-dependent oxyprolineases - OplA and OplB - was developed. Strong activity was detected on δ-valerolactam and its derivatives in the presence of ATP excess, while trace activity was detected on γ-butyrolactam. The ATP recovery system based on inexpensive Raham salt (sodium metaphosphate) and polyphosphokinase allows the use of catalytic amounts of ATP, resulting in complete transformation of 10 mM δ-valerolactam in aqueous medium at 30°C. Further improvements have been achieved by co-expressing OplA and OplB with the pETDuet1 vector, a strategy that improves soluble expression yields and protein stability. Finally, a series of phosphorylated donors in place of ATP were studied. The use of acetyl phosphate and carbamoyl phosphate with a turnover of up to 176 provides hints of possible mechanisms.
- 1. Expression of PpOplA and PpOplB alone and activity assays
The synthetic genes encoding PpOplA and PpOlpB are sequenced and cloned in the vector pET28a(+), flanked by the N-terminal restriction site NdeI and the C-terminus HindIII, as well as codons optimized for expression in E. coli. Heterologous overexpression of two proteins carrying N-terminal His-tags resulted in poor soluble expression at 30°C induced by isopropyl β-d-1-thiogalactopyranoside (IPTG) in E. coli BL21 (DE3) in lysogenic broth, similar to the results of the test at lower temperatures (20 °C). Therefore, the authors chose to express under auto-induced conditions. The presence of ammonium bicarbonate in the lysis buffer during sonicated cell lysis is critical for the release of soluble proteins, especially if PpOplA is expressed, which typically exhibits low soluble expression or low soluble protein release. Since the amount of PpOplB obtained after purification by immobilized metal affinity chromatography is very small, biotransformation is first performed by mixing two cell-free extracts (CFEs) that are obtained by sonication of the two supernatants following cell lysis.
The first test was performed in an aqueous solution of ammonium bicarbonate (50 mM, pH 8.5) at 30 °C at 1 eq. ATP and equal amounts of total protein PpOplA and PpOplB were performed in the presence of CFEs for 16 h. After derivatization with ethyl chloroformate, the formation of 5-aminovaleric acid (2b) was monitored by gas chromatography and a conversion rate of 70% was reached, indicating that PpOplA/PpOplB pairs are highly active in 1b hydrolysis (Table 1, entry 2). In the presence of excess ATP, complete transformation can be obtained (2.5 eqATP., Table 1, entry 3). Under these conditions, γ-butyrolactam (1A) is undetectable for product formation. The use of a large excess of ATP was found to be detrimental to the reaction to 1b, as the conversion rate dropped to 35% when using 12.5 mM of ATP. Conversion rates of up to 80% can be achieved at 5 mM 1b and 7.5 mM ATP (Table 1, entry 6). Under the test conditions, no transformation was observed at 10 mM 1b. The conversion of ε-caprolactam (1c) remained low at both 2 mM and 5 mM substrate concentrations (25% maximum, Table 1, entries 9 and 10), highlighting the PpOplA/PpOplB preference for 6-membered cyclic lactams. In the absence of ATP, PpOplA, PpOplB, or the presence of both PpOplA and PpOplB, different control reactions are performed in parallel; No amino acids were detected in any of these control groups. When the reaction was performed in potassium hydrogen phosphate buffer (50 mM, pH 7.5) in the absence of ammonium bicarbonate, no product was detected, confirming past observations using homologous PjCapAB that bicarbonate is critical for activity. It is important to note that enzyme preparations have significantly reduced activity after freeze-thaw, so prepare fresh CFE prior to each round of biotransformation. Lyophilized whole-cell mixtures expressing PpOplA and PpOplB, respectively, were found to be active in the hydrolysis of 2 mM 1b and 5 mM ATP, but achieved lower conversion rates (maximum 50% conversion rate). The limitation in the use of whole-cell mixtures may be due to the necessary transfer from the first intermediate obtained with PpOplA to the second biocatalyst preparation, which requires exchange from one cell to another. Due to the low amount of PpOplB recovered, the use of purified protein was compromised, and the first attempt did not result in any transformation. Early studies have shown that fragment B purified from Pseudomonas putida has a tendency to aggregate, affecting activity. Given the negative effects of high concentrations of ATP on conversion rates (see Table 1), a stepwise addition of ATP was implemented. The addition of three batches of 5 mM ATP over 2 hours resulted in a large increase in the formation of 2b (up to >99% conversion from 6 mM 1b and 6.9 mM 2b from 8 mM 1b, Table 2, entries 1 and 2). At 1c, the effect remained modest (up to 1.4 mM2c, Table 2, entries 4-6), confirming the preference of the PpOplAB system for the valerolactam ring under these reaction conditions.
Figure 1
Table I
Table II
- 2. Co-expression of PpOplA and PpOplB in the pETDuet1 vector
Given the poor stability of enzyme preparations during storage and the low soluble expression of PpOplB, improved protein expression is needed to further investigate catalytic activity. The decision to co-express PpOplA and PpOplB using a co-expression vector, taking into account the crystal structure of the homologous caprolactamase system from Pseudomonas jasonii revealed heterotetrameric assembly, in which each CapB subunit interacts with the CapA subunit. Co-expression of two subunits of the OplAB system within the same cell is expected to favor the formation of tetrameric assembly upon protein expression, which may have a positive effect on the stability of the overall assembly. In addition, this protocol greatly simplifies the preparation of biocatalysts. Co-expression of both enzymes in E. coli BL21 (DE3) was successfully co-induced in an excellent broth at 24°C using a pETDuet1 vector with two multiple cloning sites. Of note, there was a significant increase in the soluble expression of PpOplB and comparable yields of soluble proteins between PpOplA and PpOplB (Figure 3). This method allows for the generation of stable assemblies in solution, as evidenced by the successful co-purification of the two proteins by IMAC. Since only PpOplA carries a His-tag at the N-terminus, this indicates a strong interaction between the two subunits and the formation of protein complexes. The 1:1 molar yield of the soluble assembly was confirmed by optical density analysis by SDS-PAGE. The presence of bicarbonate in the lysis buffer helped to increase protein yield, but in this case it was found that a good release of soluble protein was not mandatory, and yields of both proteins were also obtained in Tris and HEPES buffers.
The activity of co-expressed PpOplA and PpOplB (now known as PpOplAB) was investigated on a range of lactams (Figure 2). To simplify analysis and avoid time-consuming derivatization of GC analysis, substrates and products are co-analyzed directly from aqueous reaction mixtures by LC-MS. In the presence of 12.5 mM ATP, PpOplAB was used as CFE for 10 mM 1a-h (total protein content of 8 mg/mL), corresponding to ∼7 μM PpOplAB based on optical density analysis. The strongest activity was observed at 1b (complete depletion) and 5-methylpiperidin-2-one (1f, resulting in a 7.9 mM product, Table 3), while transformation remained modest at 1c (formation of 2.3 mM2c, Table 3, entry 3). For the first time, some products were observed in the transformation of γ-butyrolactam (1a), corresponding to ∼4% substrate consumption. For other methyl derivatives of δ-valerolactam, the conversion was moderate (maximum 2.4 mM product, Table 3), and the substitution closest to the amide bond (especially the 3 and 6 positions) had the greatest negative impact on the conversion. In contrast, the furthest carbonyl substitution has little effect on activity, suggesting that there may be steric hindrance near the reactive moieties of 1d, 1e, and 1g. Finally, 3-ethoxycarbonyl-2-piperidone (1H), which has a large electron-withdrawing substituent at the α position of the carbonyl group, is not accepted. All substituted lactams were tested as racemic mixtures. Strong activity against 1F has been implied with poor enantioselectivity (79% conversion rate). The enantioselectivity of compound 1e was further investigated because the activity was high enough to deliver the product for enantiomeric excess (EE) analysis while the conversion rate remained below 50%, which may indicate a case of kinetic resolution. A method was developed that allows the measurement of the ee value of product 2e on chiral phase HPLC after derivatization and the ee value of remaining substrate 1e on chiral phase GC. PpOplAB shows low (R) enantioselectivity and delivers (R)-2e with 68% ee and (S)-1e with 8% ee, which corresponds to an E value of 6. Overall, the data suggest that amino acids with high enantiomeric purity cannot be delivered using the kinetic resolution of PpOplAB. Notably, the tolerance of PpOplAB to high concentrations of ATP highlights the advantages of co-expression for protein stability, which may be related to stable assembly. CFEs have also been found to be insensitive to freeze-thaw (Table 3, entry 8). While PpOplAB can be purified (see Figure 3), the recovered protein has poor activity against 1b under test conditions (maximum 15% converted to 2b). Therefore, all further reactions are performed using CFE.
With a lower amount of enzyme (total protein content in 2 mg/mLCFE), the aim is to capture changes in product formation before complete transformation occurs. A clear trend was observed in the transformation of 10 mM 1b, with the highest product formation obtained at a large excess of bicarbonate (55 mM), which is consistent with the observations made with PjCapAB.
ATP concentrations were found to be closely related to the amount of product formed, and in general, some uncoupling could be expected, as the amount of 2b formed was found to be lower than the ATP concentrations used in all cases (see Table 4), and a slight excess of ATP was required to achieve the highest conversion rate.
Figure 2
Figure 3
Table III
Table IV
- 3. ATP regeneration
The reaction of OplAB to 5-oxo-l-proline requires ATP and CapAB with ε-caprolactam. An ATP-binding site was detected in PjCapA, and the associated amino acids involved in binding were conserved in PpOplA. ATP is thought to activate the substrate through phosphorylation by the action of subunit A and is released in the form of ADP, while subunit B hydrolyzes the phosphorylated species in subsequent steps. This explains the need for at least stoichiometric amounts of ATP, which is actually incompatible with atomic-economical and cost-effective biocatalytic applications. Therefore, the catalytic amount of ATP for lactam biocatalytic hydrolysis was investigated by implementing an enzymatic regeneration system based on class I polyphosphate kinase 2 (PPK2-I), which catalyzes the monophosphorylation of ADP from inexpensive inorganic polyphosphates (Figure 4). PPK2-I SMc0214828 from Sinorhizobium meliloti was selected because the enzyme has been shown to be compatible with other ATP-dependent enzyme systems. The enzyme is overexpressed in E. coli and added as CFE to a standard reaction mixture supplemented with inexpensive Reham salts (i.e., sodium metaphosphate). Complete conversion of 10 mM 1b can be observed in the presence of 0.5 mM ATP, 5 mM Graham salt, and 25 mM ammonium bicarbonate (see Table 5). In the absence of kinases, the conversion rate is strictly limited to the catalytic amount of ATP present (<5% conversion rate). The higher the polyphosphate content, the higher the conversion rate. Under the reaction conditions tested, there was a clear advantage over the addition of excess ATP, which resulted in only 31% activity obtained using the recovery system (see Table 5, entry 4). Further tests were performed and showed that 0.1 mM ATP was sufficient to achieve a full consumption of 10 mM 1b (see Table 6). Increasing the ATP concentration from 0.1 mM (0.004 eq.) to 0.5 mM (0.02 eq.) at 25 mM at 1b had a strong positive effect on the conversion rate and formed up to 13.9 mM of 2b in the presence of 10 mM Graham salt (Table 6). Temporal studies have shown that the response is particularly fast during the first 90 minutes (forming 7.9 mM of 2b) and then begins to slow down, reaching maximum conversion after about 10 hours. Increasing the substrate concentration to 50 mM will hardly improve product formation (2b at 14.7 mM), even at higher bicarbonate concentrations. Overall, the data suggest that the PpOplAB/SmPPK2 platform can generally maintain high substrate concentrations. Optical density analysis based on SDS-PAGE to estimate the amount of enzyme PpOplAB (3.4 μM) and SmPPK2 (18.1 μM) in the crude lysate. In the case of biotransformation of 50 mM 1b resulting in the formation of 14.7 mM 2b, the turnover number (TON) for PpOplAB is 4360 and the TON for SmPPK2 is 785. Both values indicate that the catalytic efficiency of δ-pentanolactam cleavage is from good to excellent under ATP regeneration conditions.
Similar to PjCapAB, PpOplAB requires bicarbonate to function. A direct correlation can be seen between the concentration of bicarbonate and the amount of product formed, with the highest product concentration obtained in case of excess bicarbonate. With PjCapAB, carboxyphosphates formed from bicarbonate and ATP are assumed to be possible intermediates capable of phosphorylating substrates in various mechanistic recommendations. Therefore, it is assumed that other phosphoric acid donors similar to carboxyphosphate can bypass the stringent requirements for ATP. A range of alternative phosphoric acid donors were explored (Figure 5) and an alternative excess ATP (1.25 eq.) was added. When using 2-phosphoenolpyruvate (3C), product formation is not detectable. In contrast, the use of carbamoyl phosphate (3a) and acetyl phosphate (3b), which are structurally associated with carboxyphosphate 3d, resulted in the formation of 0.3 mM2b, equivalent to 10% of the activity obtained with the same amount of ATP (Table 7). Thus, PpOplAB is catalytically active in the hydrolysis of 1b in the absence of ATP, and a TON of 176 is achieved using alternative phosphoric acid donors 3a and 3b. These data provide some clues that structurally associated carboxyphosphates may be involved in the first step of the reaction, and that under standard conditions, ATP may not directly phosphorylate the substrate, contrary to the hypothesis of a bacterial 5-oxoprolinease, where no bicarbonate dependence has been reported. This suggests that ATP acts with PpOplAB in a similar way to the case of acetone and acetophenone carboxylases, which produce carboxylphosphate as the first intermediate in the catalytic cycle. However, these enzymes transfer the carboxyl moiety instead of phosphate to their respective substrates. Phosphate transfer of PpOplAB to substrates may not be enzymatic, and a range of cases of spontaneous phosphorylation of receptors with acetyl phosphate have been reported. It is clear that PpOplA and other homologous subunits are necessary to allow the utilization of ATP as a primary source of phosphate during substrate activation, and the exact mechanism of this remains to be elucidated.
Figure 4
Figure 5
Table V
Table VI
Table VII