Centre for Protein Engineering, MRC, Hills Road, Cambridge CB2 2QH, UK
1 To whom correspondence should be addressed. E-mail: arf25{at}cam.ac.uk
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Abstract |
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Keywords: evolution/DAH7PS/KDO8PS/metal binding/metal-dependent
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Introduction |
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Despite numerous studies of the reaction catalysed by KDO8PS (Kohen et al., 1992; Baasov et al., 1993
; Dotson et al., 1993
; Liang et al., 1998
; Sheflyan et al., 1999
) and DAH7PS (Floss et al., 1972
; Stephens and Bauerle, 1992
; Akowski and Bauerle, 1997
), which have been centred almost exclusively on the enzymes from Escherichia coli, details pertaining to the mechanism of these enzymes and the nature of the reaction intermediates continue to remain uncertain. It is clear that both enzymes catalyse a reaction in which a carbanion equivalent generated at the C-3 on the si-side of PEP is directed to react with the re-face of the electrophilic carbonyl of the respective monosaccharide (Kohen et al., 1992
; Dotson et al., 1993
).
Until very recently, however, there was thought to be one prominent difference between KDO8PS and DAH7PS enzymesDAH7PS enzymes were known to require a metal ion for activity (Stephens and Bauerle, 1991) whereas KDO8PS enzymes do not (Ray, 1980
). It has been shown through metal analysis (Stephens and Bauerle, 1991
) and chemical modification experiments (Stephens and Bauerle, 1992
) that DAH7PS enzymes require the metal ion for catalytic activity rather than for structural integrity. Based on low sequence identity (18%) and the difference in metal requirement, the mechanistic relationship between these two enzymes has been assumed to be primarily coincidental.
This view has been challenged with the detailed study of Aquifex aeolicus KDO8PS that is found to bind and require metal ions for activity in a fashion similar to that of DAH7PS while having a high sequence identity with E.coli KDO8PS. A.aeolicus KDO8PS can bind a range of divalent metal ions including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+ and Zn2+, a property shared by other bacterial and eukariotic DAH7P synthases. The level of activation depends on the identity of the metal, with the highest activity observed with Cd2+ and Mn2+ for A.aeolicus KDO8PS (Duewel and Woodard, 2000). Later, KDO8PS enzymes from Helicobacter pylori and Chlamydia psittaci were found to bind divalent ions (Birck and Woodard, 2001
).
The discoveries of metal binding KDO8PSs have prompted the suggestions that perhaps metal requirement is not unique to the A.aeolicus KDO8PS and that possibly KDO8PS enzymes could be divided in two classes with respect to their metal-binding ability. Sequences of three known metal-binding enzymes were compared with the sequences of three non-metal-binding enzymes and nine residues that were strictly conserved within the classes but different between the classes were identified. It was proposed that those residues determine the ability of KDO8PS to bind metal (Birck and Woodard, 2001). Here we used protein engineering to analyse the possibility of converting E.coli enzyme into a metal ion-activated KDO8PS to explore the relationship between the classes.
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Materials and methods |
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Phosphoenolpyruvate mono(cyclohexylammonium) salt, D-arabinose 5-phosphate disodium salt, D-erythrose 4-phosphate sodium salt, MnCl2, CaCl2, CuSO4 and MgCl2 were purchased from Sigma. ZnCl2 was purchased from AnalaR and Co2Cl from Fisher.
Purification of KDO8PS
Aquifex aeolicus KDO8PS and E.coli KDO8PS genes were cloned into pMAL-c2 expression vector in a fusion to MalE (maltose-binding) protein. Point mutations introducing N26C and M25P N26C substitutions into E.coli KDO8PS gene were introduced using Stratagene QuikChange site-directed mutagenesis kit following the instructions of the manufacturer. Aquifex aeolicus KDO8PS, E.coli KDO8PS and N26C and M25P N26C E.coli KDO8PS mutants were overexpressed in C41 cells. KDO8PS and DAH7PS aldolase genes were overexpressed from pMAL 2c or pMAL 2p vector as a fusion protein with MalE. Protein was expressed at 37°C in E.coli strain C41 (Miroux and Walker, 1996). Freshly transformed individual colonies from TYE, 100 mg/ml ampicillin, 1% glucose plates were transferred to 5 ml of 2 x TY starter cultures containing 100 mg/ml ampicillin. These were grown at 37°C with shaking at
250 r.p.m. until A600 = 0.3 and then used as an inoculum for 1 l flasks containing 0.2 l of 2 x TY with 100 mg/ml ampicillin. Cultures were initially grown at 37°C to A600 = 0.8 before overnight induction with 1 mM IPTG in a thermostated shaker at 20°C. Cells were harvested after 14 h of expression by centrifugation at r.c.f. 12 000 g for 7 min in a Sorvall SLA-3000 rotor cooled to 4°C. Cell pellets containing overexpressed protein were resuspended in 50 ml of buffer 1 (20 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 7.4) and cracked (twice) using an Emulsiflex C5 high-pressure homogenizer (Glen Creston). Lysate was then centrifuged (Sorvall SS34 rotor) for 30 min at r.c.f. 16 000 g at 4°C. After centrifugation, the supernatant was added to 8 ml of prewashed amylose resin and mixed for 1 h. The resinlysate mixture was centrifuged for 5 min at r.c.f. 1500 g and the supernatant discarded. Unbound proteins were eluted from the resin by washing five times with 10 resin volumes of buffer 1 followed by centrifugation to remove the supernatant. After washing, the bound protein was eluted from the resin with five fractions of 3 ml each of buffer 1 containing 10 mM maltose. All steps were carried out at 4°C. The fractions were analysed by gel electrophoresis to check the protein purity and concentration. Relevant fractions were concentrated, flash frozen and stored at 80°C.
Metal-binding assay
Purified protein was treated with 10 mM EDTA for 1 h and dialysed extensively against buffer containing 20 mM Tris, pH 7.4 and 100 mM NaCl. Typical metal binding assay contained 75 mM protein and 375 mM CuSO4 (5-fold excess) in 100 mM NaCl, 20 mM Tris, pH 7.4. The spectrum of the protein alone was compared with that of the protein preincubated with 375 mM CuSO4 for 15 min.
Activity assay
KDO8PS activity was measured by a continuous spectrophotometric method (Stephens and Bauerle, 1991). The reaction mixture containing 20 mM Tris buffer (pH 7.4), different concentrations of PEP, 50 mM metal and 0.7 mM protein was equilibrated at 25°C for 2 min and the reaction initiated by addition of 10 ml of 6 mM A5P (final concentration 600 mM). Disappearance of PEP was monitored as 232 nm in 1.0 cm pathlength quartz cuvettes at 25°C. The initial reaction rate was measured and plotted against substrate (PEP) concentration to obtain kinetic parameters for the E.coli KDO8PS and metal binding mutants. The data were fitted to a MichaelisMenten model:
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Activity assay with different metals
N26C and M25P N26C KDO8PS activity was measured in a reaction mixture containing 20 mM Tris buffer (pH 7.4), saturating concentration of PEP (200 mM), A5P (600 mM), 50 mM metal and 0.7 mM protein (total reaction volume 100 ml). Protein was preincubated with PEP and metals (Co2+, Ni2+, Mn2+, Mg2+, Zo2+, Cu2+) at 25°C for 2 min and then reaction was initiated by addition of 10 ml of 6 mM A5P. Disappearance of PEP was monitored at 232 nm in 0.4 cm pathlength quartz cuvettes at 25°C. The velocity of the reaction with different metals was measured by steady-state kinetics.
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Results |
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In our effort to extend the understanding of metal-binding ability in the KDO8PS family of enzymes, we compared the metal-binding residues of A.aeolicus KDO8PS with the structurally corresponding residues of E.coli KDO8PS. We used ß-strands of the barrel to superpose the two proteins. We found that two out of the four residues involved in metal binding in A.aeolicus KDO8PS were structurally conserved in E.coli KDO8PS (His185, Glu222 and His202, Glu239, respectively; Figure 1). The region of the third residue, Asp233 in A.aeolicus KDO8PS, could not be assigned in the crystal structure of E.coli KDO8PS. Structure-based sequence alignment of A.aeolicus KDO8PS and E.coli KDO8PS does suggest a conservation, however (Asp233 and Asp251, respectively; Figure 2). The fourth residue implicated in metal binding, Cys11 in A.aeolicus KDO8PS, is substituted for an Asn in the E.coli enzyme. The Lys46 residue that coordinates water molecules to the metal ion in A.aeolicus KDO8PS is also conserved in E.coli KDO8PS protein (Lys61).
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The binding of Cu2+ ions to KDO8PS enzymes can be monitored by measuring the increase in absorbance at 375 nm upon CuSO4 binding due to a ligand to Cu2+ charge transfer (Solomon et al., 1993). We observed an increase in A375 nm on addition of CuSO4 in A.aeolicus KDO8PS (intensity increasing with increased concentration of metal), confirming the previously reported metal-binding ability of this enzyme (Figure 5A). We did not observe the appearance of the peak in E.coli KDO8PS upon addition of CuSO4, indicating lack of metal binding (Figure 5B). However, the mutation N26C in E.coli KDO8PS protein results in the appearance of a strong absorbance at 375 nm, indicating binding of the metal. The increase in A375 nm was also observed in the M25P N26C mutant (Figure 5C and D).
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The activity of the wild-type and mutant enzymes was determined using a continuous spectrophotometric assay monitoring the disappearance of PEP at 232 nm. Steady-state kinetics of wild-type KDO8PS and the mutants was measured at different concentrations of PEP and a constant saturating concentration of A5P (600 mM). The reaction followed MichaelisMenten kinetics for wild-type and mutant enzymes. We obtained kinetic values for the N26C and M25P N26C KDO8PS mutants in the presence and absence of the metal ion. Mn2+ was used as it leads to a maximum level of activity in the metal-binding A.aeolicus KDO8PS.
Introduction of the metal-binding site into E.coli KDO8PS led to a decrease in kcat of the enzyme. We observed different properties of the metal-binding mutants in the presence and in the absence of the metal. In the presence of metal, the kcat of both mutants was greater than in the absence of metal. It was 5-fold lower than wild-type in the N26C mutant and 1.5-fold lower than wild-type in the M25P N26C double mutant. Metal binding, however, led to an increase in KM(PEP) for both mutants (Table I), which suggests that Mn2+ binds similarly to A.aeolicus KDO8PS in proximity to PEP. Overall, M25P N26C KDO8PS was a much better enzyme with kcat/KM 510 times higher than that of N26C KDO8PS and four times higher than A.aeolicus KDO8PS. It should be noted, however, that metal activation of the engineered E.coli KDO8PS (2-fold) is very modest relative to that of A.aeolicus KDO8PS (
170-fold) even if the catalytic efficiency is superior to that of wild-type A.aeolicus KDO8PS.
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Whereas the activity of the wild-type E.coli KDO8PS was not affected by the presence of different metal ions, both mutants N26C and M25P N26C KDO8PS had different levels of activity dependent on the identity of the metal. Preliminary experiments (Table II) showed that the activity of the mutants qualitatively decreases in the order Mn2+ > Mg2+ > Ni2+ = Co2+ = Ca2+ > Cu2+ > Zn2+ for both mutants, similar to the activation pattern observed for A.aeolicus KDO8PS (Mn2+ > Ni2+ = Co2+ > Ca2+ > Cu2+ > Mg2+ = Zn2+). Metals other than Mn2+ actually inhibit the engineered E.coli KDO8PS. Aquifex aeolicus KDO8PS is poorly activated by Mg2+; similarly, the activity of both N26C and M25P N26C KDO8PS in the presence of Mg2+ ions was almost equal to that in the absence of metal ions.
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Discussion |
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The value of kcat of the N26C metal-binding KDO8PS was greater in the presence of metal, but it was reduced relative to that of the wild-type E.coli KDO8PS. The increase in activity in the presence of metal is only small but it is real, as is evident from spectral studies. In the presence of the metal, KM(PEP) is increased, suggesting that metal binds in the active site, in proximity to PEP, similarly to A.aeolicus KDO8PS.
The M25P N26C KDO8PS mutant had a value of kcat comparable to that of wild-type enzyme and, similarly to the N26C mutant, kcat and KM (PEP) were greater in the presence of metal. The increase in kcat of the double mutant in the presence of metal is only small (2-fold) but greater than of a single mutant. At physiological concentrations of PEP (9.60.3 mM (Hogema et al., 1998
), both metal-binding mutants have much higher catalytic activity than the natural metal-binding KDO8PS from A.aeolicus. Mutants of E.coli KDO8PS that gained metal-binding ability are differentially affected by various divalent ions, with the highest level of activation being achieved in the presence of Mn2+ ions, followed by the activity with Mg2+ ions and in the absence of any metal. All other metal ions in fact inhibit the activity of the metal-binding mutants of E.coli KDO8PS.
Our experiments support the previously introduced hypothesis that both KDO8PS and DAH7PS enzymes may share a common ancestor (Duewel and Woodard, 2000; Birck and Woodard, 2001
) and suggest that it was a metal-dependent protein. The N26C or M25P N26C E.coli KDO8PS are probably similar to a probable evolutionary intermediate between metal-dependent and non-metal-dependent KDO8PSs, an enzyme that binds and is activated by Mn2+, but also has a considerable activity in the absence of Mn2+ ions. We can envisage that the ancestor of E.coli KDO8PS and several other KDO8PS proteins from different species have lost one of the ligands for the metal and adapted to function in the absence of the metal ions, whereas some KDO8PS enzymes, such as KDO8PS from A.aeolicus, have retained the metal-binding ability that they share with DAH7PS. Evolutionary pressure on metal-binding KDO8PS has led to their increased activation by Mn2+ and loss of activity in the absence of metal ions.
This also illustrates how evolution of an enzyme commits itself once the direction is chosenthe loss of metal binding has probably led to a series of compensatory mutations that would allow the enzyme to function better in the absence of metal. When the metal binding is reintroduced, the protein's compensatory mutations reduce it from efficient functioning in the presence of metal. Metal occupancy of the engineered site increases the activity, although to a far lesser extent than in a site of A.aeolicus KDO8PS that was evolutionary maintained for metal binding and activation.
An additional conclusion that we can draw from this work is that the metal-binding ability of a number of known but not characterized KDO8PSs can be determined by a simple sequence comparison. We propose that KDO8PS from Campylobacter jejuni, Fusobacterium nucleatum, Rickettsia conorii, Rickettsia prowazekii, Bradyrhizobium japonicum, Caulobacter crescentus, Mesorhizobium loti, Brucella melitensis, Agrobacterium tumefaciens, Sinorhizobium meliloti, Ralstonia solanecearum, Xylella fastidiosa, Coxiella burnetii, Chlamydia caviae, Chlamydia psittaci, Chlamydia muridarum, Chlamydia trachomatis and Chlamydia pneumoniae are metalloenzymes and KDO8PSs from Pasteurella multocida, Haemophilus influenzae, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Salmonella typhimurium, Yersinia pestis, Pseudomonas aeruginosa, Pseudomonas syringae, Shewanella oneidensis, Neisseria meningitidis, Pseudomonas putida, Arabidopsis thaliana, Pisum sativum, Actinobacillus pleuropneumoniae, Mannheimia haemolitica and Pasteurella trehalosi are non-metal binding enzymes based on their sequence comparison and preservation of all metal-binding residues. Both distance matrix and maximum parsimony methods group the predicted metal-binding enzymes together (Figure 6A and B), separately from non-metal-binding proteins.
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References |
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Received April 29, 2004; accepted May 14, 2004.
Edited by Greg Winter
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