Reconstruction of diaminopimelic acid biosynthesis allows characterisation of Mycobacterium tuberculosis N-succinyl-L,L-diaminopimelic acid desuccinylase

With the increased incidence of tuberculosis (TB) caused by Mycobacterium tuberculosis there is an urgent need for new and better anti-tubercular drugs. N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a key enzyme in the succinylase pathway for the biosynthesis of meso-diaminopimelic acid (meso-DAP) and L-lysine. DapE is a zinc containing metallohydrolase which hydrolyses N-succinyl L,L diaminopimelic acid (L,L-NSDAP) to L,L-diaminopimelic acid (L,L-DAP) and succinate. M. tuberculosis DapE (MtDapE) was cloned, over-expressed and purified as an N-terminal hexahistidine ((His)6) tagged fusion containing one zinc ion per DapE monomer. We redesigned the DAP synthetic pathway to generate L,L-NSDAP and other L,L-NSDAP derivatives and have characterised MtDapE with these substrates. In contrast to its other Gram negative homologues, the MtDapE was insensitive to inhibition by L-captopril which we show is consistent with novel mycobacterial alterations in the binding site of this drug.

. Synthesis and detection of L,L-NSDAP. (a) Synthesis: meso-DAP is oxidised with NADP + to L-THDP by Cgmeso-DAP dehydrogenase, yielding an increase in absorbance at 340 nm. L-THDP is succinylated with succinyl-CoA (or acylated by an alternative acyl-CoA) by EcDapD to yield NS-AKP which is amidated with glutamate to form L,L-NSDAP or its acyl derivative by EcArgD. Reaction condition 1 (see text) is performed in this way. Reaction condition 2 (see text) recycles the NADPH and ammonia produced by Cgmeso-DAP dehydrogenase with glutamate dehydrogenase (green broken line). (b) Detection: L,L-NSDAP is cleaved by MtDapE to succinate and L,L-DAP which is epimerised by BaDapF and reduced by Cgmeso-DAP dehydrogenase with a concomitant increase in absorbance at 340 nm.
Scientific RepoRts | 6:23191 | DOI: 10.1038/srep23191 similarly purified to electrophoretic homogeneity as judged by SDS-PAGE (Supplementary Fig. S1b and S1c). EcArgD and EcDapD were also purified to apparent homogeneity (Supplementary Fig. S1d and S1e) and yields of about 10 mg of EcArgD and 15 mg of EcDapD were obtained per litre of culture.
Metal content and dependence of MtDapE. DapE homologues from other organisms require a tightly bound zinc ion for catalysis [9][10][11][12][13] . To determine whether MtDapE was similar in this regard, we removed zinc from pure MtDapE by dialysis against EDTA and 1, 10-phenanthroline. This resulted in complete loss of MtDapE activity. We then analysed the untreated and metal chelator treated MtDapE by inductively coupled plasma optical emission spectroscopy (Methods section). The treated enzyme contained no detectable Zn 2+ . In contrast, untreated MtDapE contained 0.8 ± 0.1 Zn 2+ per monomer and was enzymatically active.
DapE proteins when fully substituted contain two zinc/monomer 9,10,20 . However, there is little difference in catalysis incurred on the loss of a single zinc atom from the active site [10][11][12]15 . This has led to postulation of a catalytic mechanism, wherein histidine 349 of HiDapE (H330 in MtDapE) takes on the role of the second zinc ion in correctly orientating L,L-NSDAP within the active site 9 . Our observation of the activity of a singly zinc-substituted MtDapE is consistent with the operation of a similar His-dependent mechanism in this mycobacterial enzyme.
Spectrophotometric assay for an enzymatic synthesis of L,L-NSDAP, L,L-NGDAP and other DAP derivatives. In order to characterise MtDapE, it was necessary to devise a convenient method for the synthesis of its substrate. We anticipated we could reverse the DAP dehydrogenase step, generating L-THDP from meso-DAP and NADP + with E. coli DapD, succinyl-CoA (or other acyl-CoAs), E. coli DapC or its orthologue E. coli ArgD and glutamate, which would generate L,L-NSDAP, or acyl analogues thereof (Fig. 1a). Furthermore, we reasoned that we could follow this process by the addition of MtDapE and BaDapF allowing regeneration of meso-DAP which could be detected by the Cgmeso-DAP dehydrogenase originally present for the initial formation of L-THDP (Fig. 1b).
In these initial spectrophotometric experiments, we maintained the succinyl-CoA substrate of EcDapD at a concentration equal to that of meso-DAP to ensure a single turnover of the DAP added (detailed Fig. 2 legend). We were then able to monitor the synthesis of L,L-NSDAP as a function of NADPH synthesis with a concomitant increase in absorbance at 340 nm (Fig. 2a).
Consistent with this pathway, a jump in absorbance at 340 nm occurred on the addition of meso-DAP consistent with the complete oxidation of the latter. No further increase in absorbance was detected on addition of EcDapD or EcArgD until BaDapF was added when there was an additional increase in absorbance equal to that on the addition of meso-DAP (Fig. 2a). This second increase in absorbance was dependent on EcArgD, EcDapD, BaDapF, succinyl-CoA and meso-DAP and was consistent with the formation of L,L-NSDAP and its consumption by DapE.
To test the flexibility of this system with respect to generation of acyl-L,L-DAP analogues of L,L-NSDAP, we repeated the above pathway reconstruction assays replacing succinyl-CoA with glutaryl, malonyl, butyryl, acetoacetyl, acetyl, and propionyl-CoA thiol esters ( Fig. 2b-g). Glutaryl-CoA supported synthesis of a MtDapE active substrate almost as efficiently as succinyl-CoA (Fig. 2b). Far smaller or negligible quantities of product were generated with the other acyl-CoA thiol esters within the time frame of the experiment ( Fig. 2c-g).
We therefore tested the possibility that malonyl, butyryl, acetoacetyl, acetyl or propionyl-CoA thiol esters could not support the EcDapD activity efficiently enough to generate detectable quantities of acyl-L,L-DAP analogues over duration of the assay. Therefore, we assayed EcDapD and L-THDP dependent acyl-CoA deacylation via reduction of 5,5′ dithiobis(2-nitrobenzoic acid) by CoA thiol to para-thionitrobenzoate at 412 nm 21 (Methods section; Supplementary Fig. S2a,S2b).
These results were consistent with the crystal structure of the MtDapD: succinyl-CoA complex where a correctly sited carboxyl of the succinyl-CoA substrate is required to form electrostatic and hydrogen bonding interactions with MtDapD 21 .
Enzymatic synthesis, purification and characterisation of L,L-NSDAP, L,L-NGDAP and other DAP derivatives. To synthesise MtDapE substrates preparatively, we reconfigured the spectrophotometric assay of L,L-acyl-DAP synthesis as a single pot method using EcArgD, EcDapD and Cgmeso-DAP dehydrogenase (Methods section; Fig. 1a, reaction condition 1). Additionally, in an alternate reaction scheme (Fig. 1a, green dotted line) NADPH generated by Cgmeso-DAP dehydrogenase could be continuously reconverted to the starting NADP + by L-glutamate dehydrogenase which consumed the 2-oxoglutarate generated by EcArgD and the NADPH and ammonia generated by Cgmeso-DAP dehydrogenase (Methods section; reaction condition 2). This reduced the amount of nicotinamide cofactor used in the synthesis, making purification of the desired products easier. Similar strategies have been employed for synthesis of peptidoglycan precursors 22 .
We  (Fig. 3a). As confirmed by TLC, L,L-NSDAP obtained from reaction condition 1 was highly pure (Fig. 3c). No degradation of L,L-NSDAP was observed even after two years of storage at − 80 °C.
The ability to generate the MtDapE substrate lent itself to the synthesis of a number of acyl-analogues of L,L-NSDAP that we could use to probe the substrate specificity of MtDapE. We therefore pursued the synthesis of a number of L,L-acyl-DAP derivatives by varying the coenzyme A acyl donor utilised by DapD. However, cognisant of the negligible yields of acyl-L,L-DAP species afforded by most of the acyl-CoA species utilised in Fig. 2, we extended the incubation time to overnight to maximise the possibility of synthesising products from even the acyl-CoA donors that were least reactive in the spectrophotometric assay of enzymatic synthesis of N-L,L-acyl-DAP derivatives (Fig. 2d,f).
Following purification of the products of the attempted syntheses of the various N-L,L-acyl-DAP derivatives, using DapE assays to follow elution of N-L,L-acyl-DAP species, we were able to confirm the synthesis of L,L-N-glutaryl-DAP (L,L-NG-DAP) enzymatically in 30.1% yield. TLC analysis of this species suggested it was essentially homogeneous (Fig. 3c). Negative ion nanospray-MS analysis (  Apart from L,L-NSDAP and L,L-NGDAP, of the acyl-DAP species whose synthesis could be confirmed by mass spectrometry, only the synthesis designed to generate acetoacetyl-L,L-DAP produced sufficient material to quantitate gravimetrically (5.56 μmols, equating to an overall yield of 22.2%). However, here, the lack of purity of the acetoacetyl-L,L-DAP product suggested by its mass spectral analysis indicated the true yield of this DAP-derivative was considerably less than that suggested by weight. Clearly, the current methodology did not lend itself to the generation of significant quantities of the remaining acyl-L,L-DAP species whose synthesis was attempted. This was in large part due to the restrictive substrate specificity of EcDapD.
Although synthesis of the L,L-N-malonyl, butyryl, acetoacetyl, acetyl and pyopionyl-DAP species was attempted, the yields were low enough to preclude their characterization as DapE substrates. Therefore, as a preparative technique our enzymatic approach lacked the flexibility of chemical syntheses that have generated L,L-N-acetyl-DAP, L,L-N-butyryl-DAP, and L,L-NGDAP 23,24 . It does however suggest that this method could generate possibly larger analogues of L,L-NSDAP such as L,L-NG-DAP and L,L-N-pimeloyl-DAP, as well as a range of NMR-active or radio labelled L,L-NSDAP species in laboratories unequipped for stereo-chemically controlled organic synthesis required to access DAP analogues 4 .
Kinetic characterisation of MtDapE. Having achieved a viable synthesis of L,L-NSDAP, we then kinetically characterised MtDapE where its production of L,L-DAP from L,L-NSDAP could be monitored at 340 nm by coupling the enzyme to BaDapF and Cgmeso-DAP dehydrogenase catalysed reduction of NADP + (Methods section). L,L-DAP production was dependent upon MtDapE and L,L-NSDAP (Fig. 4). Progress curves (Fig. 4a) of MtDapE showed a steady state preceded by a short lag of 10 to 20 seconds. The end point of the assay indicated almost complete consumption of the L,L-NSDAP in the assay. The relationship between DapE concentration and rate was strictly linear (Fig. 4b), indicating the assay quantitatively reported DapE activity.
The dependence of MtDapE activity on L,L-NSDAP concentration was hyperbolic and could be fitted to the Michaelis Menten equation (Fig. 4d) where the MtDapE K m , k cat and k cat /K m ratio for L,L-NSDAP were 31.09 ± 3.71 μM, 4.85 ± 0.15 s −1 and 0.156μM.s respectively. This latter value is similar to that of E. coli DapE enzyme 25 suggesting that these DapE homologues had similar catalytic efficiencies.
The temperature and pH optima for DapE catalysis were determined. The temperature optimum of the reaction at pH 8.0 was between 37 to 42 °C ( Supplementary Fig. S3a). The relationship between MtDapE activity and pH was bell-shaped with a pH optimum of 7.5 ( Supplementary Fig. S3b). To ensure that this reflected MtDapE activity, the experiment was repeated at four-fold higher Cgmeso-DAP dehydrogenase or BaDapF concentrations (7.96 μM and 91.2 μM respectively), with similar results (Supplementary Fig. S3c and S3d). This suggested that the pH profile in Supplementary Fig. S3b reported the impact of pH on MtDapE activity.
The pH profile of MtDapE resembled that of HiDapE 12 . The half maximal values of the ascending limb of the pH profile for MtDapE was 6.7 ( Supplementary Fig. S3b). This value could represent dissociation of the zinc-activated water molecule central to the DapE mechanism 12 or the dissociation of H352 in MtDapE whose corresponding residue in the mono-zinc substituted HiDapE crystal structure (H349) has been implicated in orientating L,L-NSDAP within the active site 9 . The half maximal value of the descending limb of the pH profile for MtDapE was 8.2 ( Supplementary Fig. S3b). This could relate to dissociation of MtDapE amino acids or the free amino group of L,L-NSDAP or both 12 .
In order to probe the substrate structure-activity relationship of MtDapE, we sought to synthesise and test other acyl-L,L-DAP species. In this regard, MtDapE utilised L,L-NG-DAP as a substrate. We established that the assay utilising L,L-NG-DAP was linearly dependent on MtDapE protein (Fig. 4c) and found MtDapE to have a K m for L,L-NGDAP of 1024 ± 645μM and a k cat of 5.60 ± 2.88 s −1 where the k cat /K m ratio was 0.00547μM.s (Fig. 4d). The imprecision of these constants relates to the very high K m for L,L-NGDAP. Nevertheless on comparison of k cat /K m ratios, L,L-NGDAP was 28.5-fold less efficient as a substrate than L,L-NSDAP.
Hlaváček et al. 23 reported that L,L-NGDAP did not interact with HiDapE. The disparity between our data and this observation may be a species discrepancy in DapE specificity. However, Hlaváčeket al. 23 employed a DapE assay that followed the loss of absorbance due to hydrolysis of L,L-NSDAP (ε 225 nm = 698 M −1 .cm −1 ), a procedure that is one ninth as sensitive as the NADPH-coupled assay (ε 340 nm = 6220 M −1 .cm −1 ) employed here.

Response of MtDapE to inhibitors.
The assay we developed using our in situ substrate synthesis would be of utility for detection of DapE inhibitors which could potentially have antimicrobial properties. Thiols such as L-captopril are potent inhibitors of HiDapE 14 and NmDapE 10 (K i values 2.8 μM and 1.8 μM respectively). This potency stems partly from co-ordination of the L-captopril thiol between the two zinc atoms in the DapE active site 10 . Therefore to extend these studies to MtDapE, we pre-incubated L-captopril and the HiDapE thiol-inhibitors L-penicillamine 14 and 2-thiopheneboronic acid 14 with MtDapE and 31 μM of NS-DAP (the K m for this substrate) to determine the impact of these inhibitors on MtDapE.
L-Captopril at 10, 3, and 1 mM exerted 99.96%, 26.67% and 23.34% inhibition of MtDapE activity. This inhibition was considerably less than observed for HiDapE 14 and NmDapE 10 . 1 mM L-penicillamine exerted 79.41% inhibition of MtDapE which assuming the competitive kinetics displayed by HiDapE 14 , suggested that the MtDapE K i for this compound would be 28.2 fold greater than that of the HiDapE 14 . 2 thiopheneboronic acid was similarly far less potent an inhibitor of MtDapE (22.3% at 10 mM) than of the HiDapE 14 . Assuming this inhibitor behaved towards MtDapE in a non-competitive manner as it did towards HiDapE 14 , this degree of inhibition suggested MtDapE would be 515-fold less sensitive to 2-thiopheneboronic acid than HiDapE 14 .
The unreactivity of inhibitors such as L-captopril towards MtDapE was surprising. We could only detect a single zinc ion in the active site of MtDapE. The structure of the L-captopril-inhibited Neisseria enzyme revealed the thiol of the inhibitor is sandwiched between two zinc ions 10 although the loss of one zinc ion does not modify the sensitivity of Salmonella enterica DapE to L-captopril 15 . It was therefore unlikely that the MtDapE was rendered insensitive to L-captopril due to the presence of a single zinc within the active site.
On inspection of the crystal structure of the L-captopril complex with NmDapE 10 , N346, G325, Y198 and R179 interact with L-captopril. Sequence alignments (ClustalΩ 26 , Supplementary Fig. S4) of the MtDapE with other DapE homologues reveal that these residues are only completely conserved amongst Gram negative organisms. In contrast, actinomycetes including the mycobacteria have substituted NmDapE residues N346, G325, Y198 and R179 with aspartate, tryptophan, arginine and cysteine respectively (Supplementary Fig. S4). These substitutions probably underpin the loss of L-captopril potency towards MtDapE 10 .
The insensitivity of mycobacterial DapE to L-captopril and other DapE inhibitors underscore the requirement for the development of novel anti-tubercular drugs. Here, we developed a cheap and efficient method to access the L,L-NSDAP substrate of Mt-DapE that may support future screening for new DapE-directed antimicrobials.

Methods
Chemicals, strains and constructs. All the plasmids used in this study are listed in Supplementary   Table S1. Restriction endonuclease and other enzymes used for cloning were from New England Biolabs (NEB). Complete EDTA-free protease inhibitor cocktail tablets were from Roche Diagnostics, Germany. Oligonucleotides were from MWG Biotech, Germany. Succinyl-CoA was prepared as in 27 . meso-DAP was purified according to 28 . NADP + was from Melford, U.K. All other chemicals used in this study were purchased from Sigma Aldrich. Cellulose TLC plates were from Merck, Darmstadt, Germany.
E. coli BL21 (DE3) (Novagen) and C41 (DE3) 29 was used for expression. The construct of N-terminal His-tagged EcDapD in pFO4 30 was used in this study. Expression constructs of Corynebacterium glutamicum meso-DAP dehydrogenase (Cgmeso-DAP dehydrogenase) in pET28b 31 and Bacillus anthracis DapF (BaDapF) in pET23a 32 were kind gifts from Dr. David Roper, University of Warwick, Coventry, U.K.  Supplementary Table S1. MtdapE and EcargD PCR products were digested with Nde I and Hind III, ligated into similarly digested pET28b vector and transformed into E. coli Top10 competent cells. Plasmid DNA was isolated from overnight Luria Bertani (LB) cultures of single transformants grown in the presence of 25 μg/ml kanamycin. The nucleotide sequence of both constructs in frame with a 5′ sequence encoding a (His) 6 were confirmed by sequencing.

Construction of MtdapE and EcargD
Over expression of (His) 6 tagged recombinant proteins. The expression constructs pET28b-MtDapE and pET28b-EcArgD encoding MtDapE and EcArgD were transformed into E. coli C41 (DE3). A single transformant was inoculated into a starter culture of LB broth containing 25 μg/ml kanamycin and grown overnight at 37°C. A 1% (v/v) inoculum of the starter culture was added per litre of terrific broth (MtDapE) or LB (EcArgD) and cultures were grown in the presence of 25 μg/ml kanamycin at 37⁰C until an A 600 of 0.6 was reached. The cultures were then cooled to 16 °C and protein expression was induced with 1 mM isopropyl β -D-1-thiogalactopyranoside (IPTG) and incubation was continued at 16 °C for 20 hours. The cultures were harvested and cell pellets were stored at − 80 °C.
pFO4-EcDapD showed maximal expression of E. coli DapD in BL21 (DE3) cells when induced with 1 mM IPTG at 16 °C for 20 hours. The pET23a-BaDapF and pET28b-Cgmeso-DAP dehydrogenase constructs were transformed into E coli BL21 (DE3) cells and showed maximum expression of Cgmeso-DAP dehydrogenase and BaDapF when grown for 4 to 5 hours at 37 °C after induction with 1 mM IPTG. Five one litre cultures of BaDapF and two one litre cultures of Cgmeso-DAP dehydrogenase and EcDapD were grown to obtain sufficient quantities of protein after purification.
Purification of (His) 6 tagged recombinant proteins. The cell pellets of MtDapE were resuspended in Buffer A (25 mM N-(2-hydroxyethyl) piperazine-N′ -(2-ethanesulphonic acid) (HEPES) pH 7.5, 10% (v/v) glycerol, 50 mM imidazole and 1 mM dithiothreitol (DTT)) supplemented with complete EDTA-free protease inhibitor cocktail and were lysed by sonication at 4 °C with eight 30 seconds pulses interspersed by cooling for 30 seconds. The insoluble pellet was removed by centrifugation at 4 °C and 27,000 × g for 45 minutes. The soluble fraction was purified using a Ni 2+ -loaded HisTrap high performance affinity column (GE Healthcare) which was pre-equilibrated and washed with Buffer A minus the protease inhibitor and eluted isocratically with Buffer A containing 200 mM and then 400 mM imidazole. The purity of the protein was assessed by SDS-PAGE. Fractions containing MtDapE were pooled and dialysed thrice at 4 °C against 2 litres of buffer B (25 mM HEPES pH 7.5, 50% (v/v) glycerol and 1 mM DTT) and the protein was stored in − 80 °C.
The same purification protocol was followed for EcArgD and EcDapD except that Buffer A was replaced with Buffer C (20 mM Tris.HCl pH 8.0, 0.5 M NaCl, 10% (v/v) glycerol and 1 mM DTT) and the proteins were eluted with a gradient of 5-500 mM imidazole in Buffer C. Fractions were analysed by 10% SDS-PAGE and the pure proteins were pooled and dialysed three times against 2 litres of Buffer D (20 mM Tris.HCl pH 8.0, 10% (v/v) glycerol and 1 mM DTT). The EcArgD and EcDapD proteins were then concentrated and stored at − 80 °C.
BaDapF was purified essentially as described for MtDapE except that Buffer E (50 mM sodium phosphate pH 8.0, 300 mM NaCl, 5 mM imidazole and 1 mM DTT) replaced Buffer A. The column was washed with Buffer E containing 10 mM imidazole followed by buffer E containing 50 mM imidazole and then 250 mM imidazole. The fractions were dialysed against Buffer F (20 mM Tris.HCl pH 8.0, 150 mM NaCl, 5 mM DTT and 50% (v/v) glycerol).
Scientific RepoRts | 6:23191 | DOI: 10.1038/srep23191 Cgmeso-DAP dehydrogenase was purified using a HiTrap Q-Sepharose fast flow anion exchange column (GE Healthcare) which was equilibrated and washed with Buffer G (20 mM Tris.HCl pH 8.0 and 1 mM DTT) and eluted isocratically with steps of 350, 400, 450, 500 and 1M NaCl in Buffer G. Fractions containing Cgmeso-DAP dehydrogenase on SDS PAGE were pooled and dialysed against Buffer H containing 20 mM Tris.HCl pH 8.0, 50% (v/v) glycerol and 1 mM DTT and stored at − 80 °C. The ultrafiltrates were separated by anion exchange chromatography using a 40 mL Q Sepharose Source 30Q column (Amersham Biosciences), which was equilibrated with 10 mM ammonium acetate pH 7.6. The samples were diluted 20 fold to 40 ml with 10 mM ammonium acetate pH 7.6 and loaded onto the column, washed with five column volumes of 10 mM ammonium acetate pH 7.6 and eluted with fifteen column volumes of an increasing gradient of 10 to 1000 mM ammonium acetate buffer pH 7.6 at a flow rate of 10 ml/min. Chromatography was followed at 215 nm and fractions were also screened for their respective DAP derivatives by enzymatic assay (below), pooled and lyophilized three times to remove ammonium acetate. MtDapE coupled assay. The purified L,L-NSDAP and derivatives thereof were tested as a substrate in an enzyme assay for MtDapE coupled to DapF. The activity of MtDapE enzyme was measured at 37 °C by following the production of NADPH at 340 nm. Unless indicated otherwise, the standard assay consisted of 20 mM Tricine pH 8.0, 10 mM MgCl 2 , 0.6 mM NADP + , 31 μM L,L-NSDAP, 1.99 μM Cgmeso-DAP dehydrogenase, 22.8 μM BaDapF and 0.134 μM MtDapE in a total reaction volume of 200 μl. The reaction was initiated by L,L-NSDAP after a 60 to 90 second preincubation of other reaction components and was monitored for 10 minutes. Initial rates were converted to MtDapE activity (min −1 ) assuming a molar extinction coefficient of NADPH of 6220 M −1 . cm −1 at 340 nm.

Preparation of metal free-MtDapE and inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis. Metal free MtDapE was prepared by extensive dialysis for three days at 4 °C in
For characterization of the kinetics of MtDapE substrate utilization, initial rates of MtDapE catalysis were obtained between 10 μM and 320 μM acyl DAP substrate. K m and k cat values were extracted from fitting initial rate data to the Michaelis Menten equation with Graphpad prism 5 by non-linear regression. Further characterisation of the temperature and pH optima of the MtDapE enzyme were carried out with the substrate L,L-NSDAP. To determine the optimum assay temperature for activity the reaction at pH 8.0 was carried out at 25 °C, 30 °C, 37 °C, 42 °C, 45 °C and 50 °C. The impact of pH on activity was examined at 37 °C in the pH range 6.0 to 9.0 with 0.5 unit pH increments at the K m of L,L-NSDAP where the following buffers at 20 mM were used -Sodium acetate (pH 5.5), 2-(N-morpholinoethanesulphonic acid (MES) (pH 6), piperazine-N,N′ -bis (2-ethanesulphonic acid) (PIPES) (pH 6.5), 3-(N-morpholino) propanesulphonic acid (MOPS) (pH 7), HEPES (pH 7.5), Tricine (pH 8) and Tris (hydroxymethyl) aminomethane (Tris.HCl) (pH 8.5 and 9).