1 Department of Biochemistry, The University of Mississippi Medical Center, Jackson, MI 39216-4505 and 2 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine,St. Louis, MO 63110, USA
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Abstract |
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Keywords: cofactor/disulfide bonds/post-translational modification/protein biosynthesis/tryptophan tryptophylquinone
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Introduction |
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This study focuses on active-site residues of the MADH ß subunit, the positions of which in the crystal structure of MADH suggest important roles in determining the structure and function of the enzyme. The side chain oxygen of Thr122 is capable of participating in three hydrogen-bonding interactions in the enzyme active site. It is positioned ~3.0 Å from the C6 oxygen of TTQ, ~3.0 Å from the side chain oxygen of Asp76 and ~2.7 Å from the side chain oxygen of Tyr119 (Figure 3). The last strong hydrogen bond between Thr122 and Tyr119 is particularly interesting. Tyr119 is positioned at one end of a ß-sheet that is a part of the active site fold. At this residue the sheet consists of two strands and is highly irregular, containing two ß-bulges in tandem between Tyr119 and Ile123. The strong hydrogen bond between Thr122 and Tyr119 appears to help to stabilize this atypical orientation and in turn stabilize the active site fold. Asp76 is also capable of participating in multiple hydrogen-bonding interactions in the enzyme active site. It is positioned ~3.0 Å from the C6 oxygen of TTQ and ~3.0 Å from the side chain oxygen of Thr122. Asp32 also makes hydrogen bonds with a water network that interacts with active-site residues. To examine the importance of the side chain oxygens of Thr122, Tyr119, Asp76 and Asp32, these residues have been altered by site-directed mutagenesis. The results presented here suggest that the potentially reactive side chain oxygens of each of these four residues is critical for the stability of MADH and possibly its biosynthesis.
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Materials and methods |
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Native MADH was purified from P.denitrificans as described previously (Davidson, 1990). Site-directed mutants of MADH were expressed in Rhodobacter sphaeroides (Graichen et al., 1999
) and purified as described previously (Zhu et al., 2000b
). All reagents were obtained from Sigma or Aldrich and were used without further purification. The bacterial strains and plasmids which were used in this study are listed in Table I
.
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Site-directed mutagenesis was performed on double-stranded pMEG976 using a QuikChange Site-Directed Mutagenesis Kit (Stratagene) and two mutagenic primers following our previously described procedure (Zhu et al., 2000b). The primers used to create the desired mutations are shown in Table II
. Each mutation was confirmed by sequencing ~70 base pairs around the mutated site. Once the mutations were confirmed by DNA sequencing, the segment of mutated DNA was cloned back into the broad host range vector derived from pRK415-1 for expression in R.sphaeroides. As an additional control to insure that no second-site mutations had occurred and escaped detection, `back mutagenesis' was performed on the T122A and Y119F mutants to convert each site-directed mutant back to wild-type by site-directed mutagenesis using a primer for mutagenesis that was identical with the original wild-type sequence. Restoration of wild-type properties was used as further confirmation that the effects of the site-directed mutation were solely due to the single mutation and not an artifact of the procedure.
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The steady-state kinetic assay of MADH with phenazine ethosulfate as an electron acceptor was performed as described previously (Davidson, 1990). The assay mixture contained 16 nM MADH in 10 mM potassium phosphate, pH 7.5, 4.8 mM phenazine ethosulfate, 170 µM 2,6-dichlorophenolindophenol (DCIP) and various concentrations of methylamine. The reaction was started by addition of methylamine and the rate of DCIP reduction was monitored by the decrease in absorbance at 600 nm. Data were fitted to the equation
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Electrophoretic techniques
Whole cell extracts of R.sphaeroides expressing different site-directed mutants of MADH were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). Western blot analysis of these gels was performed with polyclonal antibodies which had been raised against the MADH ß subunit. The secondary antibody was an anti-rabbit IgG alkaline phosphatase conjugate.
Periplasmic extracts of R.sphaeroides expressing different site-directed mutants of MADH were prepared from freshly harvested cells as described previously (Davidson, 1990). These extracts were subjected to non-denaturing SDSPAGE using a 12.5% gel. The gels were then incubated overnight in a solution of 0.5 M potassium phosphate, pH 7.5, which contained an excess of methylamine and nitroblue tetrazolium.
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Results |
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When wild-type MADH is expressed in R.sphaeroides, MADH activity may be readily detected in crude cell extracts. Steady-state kinetic assays were performed of extracts of R.sphaeroides cells which expressed the MADH mutants listed in Table II. Of these, only cells which expressed the T122S MADH mutant produced readily detectable levels of MADH activity. To obtain a more sensitive measure of activity in crude extracts, cell extracts were subjected to non-denaturing SDSPAGE and the gels were developed using an activity-staining technique which is specific for MADH activity. A gel which shows the results obtained with periplasmic extracts of cells designed to express some of the Thr122 and Tyr119 mutants is presented in Figure 4
. From the corresponding gel which was stained for total protein, one can see that the amount of total protein in each sample that was loaded on the gel is approximately equal. The activity stain of the gel reveals that of the cells expressing the different Thr122 and Tyr119 mutants, only the extracts from the cells with the T122S mutant exhibited relatively normal activity. Weak but detectable MADH activity was observed in the extracts of cells expressing the Y119F mutant. Similar levels of activity were observed in the extracts of cells expressing the Y119E and Y119K mutants (not shown). In each case, the protein which exhibited a positive reaction corresponded in position approximately to that of wild-type MADH. No evidence for MADH activity was detected in the cells expressing the T122A and T122C mutants. To ensure that the loss of detectable MADH activity was truly the result of the site-directed mutation and not due to the occurrence of some unknown secondary mutation, the T122A and Y119F mutants were converted back to wild-type by a subsequent round of site-directed mutagenesis using the same procedure. These are designated A122T* and F119Y*, respectively. As can be seen in Figure 4
, these back-mutations restored the wild-type activity. Thus, the absence of activity in cells expressing T122A MADH and the large decrease in activity in cells expressing Y119F MADH, relative to cells expressing wild-type MADH, were due to the alteration of the single amino acid residues by site-directed mutagenesis.
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The loss or decrease of MADH activity in MADH mutants that was discussed above could be due to the production of an inactive protein at normal levels, decreased stability of the mutant protein or inability to synthesize the mutant protein correctly. To distinguish between these possibilities, Western blots with antibodies to the MADH ß subunit were performed to determine the approximate levels of the MADH ß subunit of each site-directed mutant in crude cell extracts (Figure 5, Table III
). Western blots of gels comparing extracts of cells designed to express Thr122 and Tyr119 mutants revealed that only the extracts from the cells with the T122S mutant contained significant levels of the MADH ß subunit. Either no MADH or levels below our limit of detection were present in the cells expressing the T122A and T122C mutants, as well as the three Tyr119 mutants. An example is shown in Figure 5
. From the corresponding gel which was stained for total protein, one can see that the amount of total protein in each sample that was loaded on the gel is approximately equal. Extracts of cells possessing pMEG975, which encodes the wild-type polyhistidine-tagged MADH (Zhu et al., 2000b
), were included as a positive control. As seen in Figure 5
, the back-mutation of Y119F to wild-type (F119Y*) restored normal levels, indicating that the mutation does affect the efficiency of MADH biosynthesis or stability, or both. For the Y119F, Y119E and Y119K mutants activity could be detected by activity stain of non-denaturing gels, but the level of protein produced was apparently below the detection level of the Western blot procedure. It should be noted that the activity stain is actually a more sensitive procedure than Western blotting since the intensity of the stain reflects the number of times the enzyme turns over. In these experiments, the gels were allowed to incubate overnight so that even if very little active enzyme were present, enough turnover could occur over time to detect activity.
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It should be noted that the relative amounts of protein estimated by immunoreactivity could be inaccurate if key haptenic sites are disrupted by the point mutations. This possibility is minimized but not eliminated by the use of polyclonal antibodies. As discussed below, the level of protein isolated from large scale preparations (30 l) of Y119F and D32N mutants is much less than what is obtained with the wild-type MADH, consistent with the results of the Western blots. Also, a small-scale preparation (2 l) of T122C MADH yielded no detectable protein, consistent with the Western blot result. Thus, the Western blots of crude extracts appear to be a reliable indicator of the relative amount of accumulation of each MADH mutant.
Purification and analysis of mutant proteins
Of the site-directed mutants of MADH that were prepared, only the T122S mutant was produced at significant levels. The T122S MADH was purified from a large-scale culture of R.sphaeroides and the yield of purified protein was ~50% of what is typically obtained for the recombinant wild-type protein. Analysis of the purfied T122S MADH revealed that its visible absorption spectrum and steady-state kinetic properties were indistinguishable from those of wild-type MADH. For the T122A and T122C mutations, there is no evidence for the presence of any enzyme, active or inactive. These results and those in Figures 4 and 5 suggest that while the very conservative substitution of Ser for Thr is tolerated, the T122A and T122C MADH mutants are not correctly synthesized and are rapidly degraded.
Extracts of cells expressing the Y119F mutation did give a weak but positive result when analyzed by activity staining (Figure 4). To determine how much protein is actually produced, cells expressing the Y119F MADH were grown on a large scale (i.e. 30 l) and the enzyme was purified. The amount of protein obtained was only ~5% of that typically obtained for the recombinant wild-type MADH. Furthermore, the activity decreased during the course of the purification. In contrast, the activity of the wild-type MADH is fairly stable during and after purification. Steady-state kinetic analysis of Y119F MADH indicated that its Km for methylamine was similar to that of wild-type MADH. Its kcat value was only ~1% of that of the wild-type. Because of the low yield of protein and its apparent instability, it was not possible to determine whether the low kcat value reflected a true decrease in activity or an apparent decrease due to the degradation of most of the enzyme leading to loss of activity during the purification. Given the very low yield of the Y119F mutant, we did not attempt large-scale purifications of any of the other Tyr119 mutants which exhibited similar low levels of activity. The wild-type MADH which was regenerated by back-mutation of Y119F (F119Y*) was also purified. It was produced in normal yields and exhibited wild-type spectral and kinetic properties. This again confirms that the dramatic decrease in yields of the site-directed mutants which was observed was due to the specific mutations and not artifacts of the mutagenesis procedure.
The D32N MADH mutant was also purified from a large-scale growth. As was observed with the Y119F mutant discussed above, the yield was only a small percentage of that normally obtained with the wild-type and the D32N MADH was also less stable. Steady-state kinetic analysis indicated that the kcat value for this mutant was only ~1% of that of the wild-type. Again, it was not possible to determine whether the low kcat value reflected a true decrease in activity or an apparent decrease due to the degradation of much of the enzyme leading to loss of activity during the purification. Interestingly, the Km value for the D32N mutant was approximately 1000-fold greater than that of wild-type MADH.
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Discussion |
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It has been shown for another quinoprotein, the topaquinone (TPQ)-dependent amine oxidase, that the post-translational insertion of two oxygen atoms into a tyrosine residue to form TPQ is an autocatalytic process (Cai and Klinman, 1994; Matsuzaki et al., 1994
). It requires only oxygen and copper. Copper is also present in the active site of the mature enzyme. However, it was shown that site-directed mutagenesis of the residue either immediately preceding or following the precursor tyrosine decreased the rate of cofactor biogenesis (Dove et al., 2000
). It was suggested that these other residues may help to position the precursor tyrosine for the first step in the biogenesis of the mature enzyme. The potential for hydrogen bonding between the O6 of TTQ and the reactive oxygens of Thr122 and Asp76 suggests the possibilities that Thr122 and Asp76 could play roles in the formation of TTQ. It is believed that TTQ biogenesis is a multi-step process that involves oxygenation of Trp57 followed by cross-linking, possibly from nucleophilic attack on the oxygenated Trp57 by Trp108 (McIntire et al., 1991
; Itoh et al., 1995). For the oxygenation reactions of Trp57, two alternative dioxo compounds are possible. In model studies of this reaction, only ~10% of the product is the 6,7-orthoquinone (i.e. the proper substrate for the subsequent cross-linking reaction) (Itoh et al., 1995). Hydrogen bonds between the O6 of the oxygenated Trp57 (Trq57) and Thr122 or Asp76 could serve to stabilize selectively the proper oxidation product during TTQ biogenesis. It could also help to position properly the oxygenated Trq57 for nucleophilic attack by Trp108 to complete TTQ formation. If TTQ were not formed as a result of the T122A and D76N mutations, this could explain the lack of any detectable activity or protein in crude extracts of cells, relative to the low but detectable levels of active protein with mutations of Tyr119.
The low levels of expression of the D32N MADH mutant are surprising since the position of Asp32 in the crystal structure suggests it to be less directly involved than Asp76, Thr122 and Y119 in interaction with TTQ or structural stabilization. While near the active site, the side chain of Asp32 forms hydrogen bonds to a water network (Chen et al., 1998). Interestingly, a side chain O of Asp32 is also within 2.6 Å of the backbone O of N104, suggesting that Asp32 is protonated and participating in a strong hydrogen bond with the polypeptide. A similar close interaction involving the residue corresponding to Asp32 is also found in MADH from Methylobacterium extorquens AM1 (Labesse et al., 1998
). The low level of expression of D32N may reflect the importance of the detailed configuration of the protein for folding. It may also mean that the low levels of expression of the Tyr119 mutants (discussed earlier) could arise from similar effects on folding rather than a more direct effect in the active site.
The results presented here suggest that two different effects are caused by the site-directed mutagenesis of active-site residues of MADH. With the exception of T122S, all mutants show a greatly reduced level of expression which may result from a reduced rate of proper folding during biosynthesis, followed by degradation of a large fraction of the expressed protein. In the case of T122A, T122C and D76N there may also be a specific inactivation of catalysis or TTQ formation. The reduced folding rate could arise from impairment of disulfide bond formation. There are six disulfide bonds in the 12.5 kDa ß subunit of MADH. The rate of formation of these disulfide bonds, which could be important for protein stabilization, may depend critically on slight perturbation of the protein conformation during folding, as caused by mutations in Tyr119, Thr122, Asp76 and Asp32. This may account for the considerably higher sequence conservation among ß subunits than among subunits of MADH. Consistent with this notion, it is noteworthy that while the previously described F55A mutation on the
subunit of MADH dramatically affected the substrate specificity, that mutant was expressed and purified from the same heterologous expression system at a level comparable to that of wild-type MADH (Zhu and Davidson, 1999
).
There is precedence for dual roles of enzyme active-site residues in addition to their participation in catalysis. As discussed earlier, it has been suggested that in the amine oxidases such residues may also be important for cofactor biogenesis. Another example is vanillyl alcohol oxidase. This enzyme contains a covalently bound FAD which is linked to the side chain N of His422. Site-directed mutagenesis studies showed that another active-site residue, Asp170, not only participated in catalysis but also was required for the autocatalytic flavination reaction (van den Heuval et al., 2000). Roles of active-site residues in protein stability have also been documented. In class A ß-lactamases, an active site serine has been proposed to participate in catalysis and also to stabilize the active site structure (Yang et al., 1999
). In the crystal structure of the protein, Ser130 appears to form a hydrogen bond with Lys234 which links two structural domains of the protein and seems to maintain the structure of the active-site cavity. Mutation of this residue to Ala or Gly yielded enzyme which retained some activity but was less stable than the wild-type enzyme (Jacob et al., 1990
). Similarly, site-directed mutagenesis studies have shown that Asp121 of ribonuclease A participates in hydrogen-bonding interactions that contribute to both catalysis and stability (Quirk et al., 1998
). Ketosteroid isomerase exhibits a hydrogen bond network in the active site which involves an aspartate and three tyrosine residues. Site-directed mutagenesis studies demonstrated that this network is needed both for catalysis and stability (Kim et al., 2000).
There are numerous examples in the literature of studies that have shown that conserved amino acid residues in a family of proteins play either an important role in stabilizing the protein structure or an important role in catalysis. There are relatively few examples, such as those discussed above, where it has been documented that a conserved amino acid residue is necessary not only for catalysis and but also for structural stabilization or biosynthesis, or both. With MADH it was not possible at this time to assess the potential roles of these active-site residues on activity since mutation of these residues so severely affects the levels of protein produced. Roles in catalysis are suggested from their positions in the crystal structure and the generally accepted chemical reaction mechanism of MADH (Chen et al., 1998; Zhu and Davidson, 1999
). Some evidence has previously been provided for roles of Tyr119 in determining the substrate specificity of MADH (Zhu et al., 2000a
) and in monovalent cation binding (Labesse et al., 1998
). When potentially reactive residues are conserved in the enzyme active site, it is usually assumed that they must be important for catalysis. However, evolution may not necessarily have produced these residues for the purpose of optimizing a catalytic function. The catalytic function may have arisen as a consequence of a set of residues present at the active site which evolved for other functions such as to stabilize the protein structure or to facilitate its biosynthesis. Superfamilies of enzymes have been characterized which share a common structural scaffold yet catalyze different reactions (Babbitt and Gerlt, 1997
). It is believed that some residues in the active sites are used to catalyze reaction steps that are common to each enzyme whereas other active-site residues have evolved to allow for different overall activities. MADH is difficult to evaluate in this context because there are no other proteins which have been identified that exhibit significant similarity that have any function other than that of an amine dehydrogenase. Furthermore, no enzymes other than MADH and the related enzyme aromatic amine dehydrogenase (Govindaraj et al., 1994
) have yet been found which also possess the TTQ prosthetic group. In MADH, the possibility that catalytic function, stability and protein and cofactor biosynthesis may be linked by multiple roles of specific conserved amino acid residues is suggested by these results. While such a linkage may not be a common feature in the evolution of all enzymes, it may be common to certain classes of enzymes such as complex redox enzymes which require post-translational modification for cofactor biosynthesis or incorporation.
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Notes |
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Acknowledgments |
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References |
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Received March 12, 2001; revised June 15, 2001; accepted June 23, 2001.