Active-site residues are critical for the folding and stability of methylamine dehydrogenase

Dapeng Sun1, Limei H. Jones1, F.Scott Mathews2 and Victor L. Davidson1,3

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of the ß subunit of MADH which are in close proximity to the tryptophan tryptophylquinone (TTQ) prosthetic group were modified. The crystal structure of MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively conservative mutations which removed the potentially reactive oxygens on the side chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly reduced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts with antisera specific for the MADH ß subunit. No activity or cross-reactive protein was detected in extracts of cells expressing D76N, T122A and T122C MADH mutants. Very low levels of active MADH were produced by cells expressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutants were purified from cell extracts and found to be significantly less stable than wild-type MADH. Only the T122S MADH mutant was produced at near wild-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH ß subunit, protein folding and TTQ biosynthesis are discussed.

Keywords: cofactor/disulfide bonds/post-translational modification/protein biosynthesis/tryptophan tryptophylquinone


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Methylamine dehydrogenase (MADH) from Paracoccus denitrificans is a periplasmic enzyme that catalyzes the oxidative deamination of methylamine to formaldehyde plus ammonia and the subsequent electron transfer to a type I copper protein, amicyanin (Davidson, 1993Go; Hartmann and McIntire, 1997Go). MADH is composed of four subunits that are arranged in a symmetrical {alpha}2ß2 structure (Chen et al., 1998Go). Each ß subunit possesses a tryptophan tryptophylquinone (TTQ) prosthetic group (McIntire et al., 1991Go) which is formed by post-translational modifications of Trp57 and Trp108 of the ß subunit. As can be seen from its structure (Figure 1Go), TTQ biosynthesis requires insertion of oxygens at the C6 and C7 positions of the indole ring of Trp57 to form Trq57 (the quinolated ring derived from Trp57) and formation of a covalent bond between the indole rings of Trp57 and Trp108. This redox-active cofactor participates in both the catalytic and electron transfer reactions of MADH.



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Fig. 1. Tryptophan tryptophylquinone (TTQ) and the active site of MADH. The two components of TTQ are indicated, Trp108 and Trq57 (the quinolated ring that was derived from Trp57). The four residues which were the subjects of the site-directed mutagenesis studies described in this paper are also shown. Relevant hydrogen bonds which are inferred from the crystal structure are indicated as dashed lines with the distance between participating oxygen atoms listed. The C6 and C7 carbonyl carbons of TTQ are also indicated.

 
In addition to TTQ, the MADH ß subunit exhibits other unusual structural features. The cysteine and proline contents of this subunit are much higher than normal. Of the 131 amino acid residues of the ß subunit, there are nine prolines, seven of which are conserved in the other four MADHs which have been sequenced. There are also 12 cysteines, all of which are conserved in the other MADHs (Figure 2Go) and which form six intrasubunit disulfide bonds (Chen et al., 1998Go). MADH exhibits strong stability against extremes of pH and temperature (Husain and Davidson, 1985Go; Davidson, 1993Go). The unusually large number of disulfide bonds in the MADH ß subunit may be at least partially responsible for the relatively strong stability of MADH by allowing the protein to maintain a relatively rigid structure. These six disulfide bonds, as well as the large number of proline residues, are also likely to be important in determining the proper tertiary structure of the MADH ß subunit.



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Fig. 2. Amino acid sequence comparison of the ß subunit of MADH from different bacteria. Sequences are numbered relative to the P.denitrificans sequence. The sequences are those of MADH from P.denitrificans (Chistoserdov et al., 1992Go), Paracoccus versutus (formerly Thiobacillus versutus) (Ubbink et al., 1991Go), Methylobacterium extorquens AM1 (Chistoserdov et al., 1991Go), Methylobacillus flagellatum KT (Gak et al., 1995Go) and Methylophilus methylotrophus W3A1 (Chistoserdov et al., 1994Go). Conserved cysteine residues which participate in six intrasubunit disulfide bonds are highlighted in yellow. The four conserved residues which were the subject of these site-directed mutagenesis studies are highlighted in red. The two tryptophan residues which are used to form TTQ are highlighted in blue.

 
Previous studies in this laboratory have allowed us to describe the kinetic and chemical reaction mechanisms of P.denitrificans MADH (Davidson et al., 1995Go; Zhu and Davidson, 1999Go). Steady-state and microscopic kinetic rate constants have been determined and several reaction intermediates have been identified. It has been proposed that multiple residues in the enzyme active site are required for catalysis in the overall reaction mechanism (Chen et al., 1998Go). Inspection of the crystal structure of MADH reveals that the protein environment which surrounds TTQ is relatively hydrophobic with 20 residues of the ß subunit located within 4.0 Å of some portion of TTQ. The reactive C6 carbonyl of TTQ is exposed in a relatively hydrophilic, but solvent-inaccessible, region of the active site with three relatively polar residues (Asp76, Thr122 and Tyr119) in close proximity to the C6 of TTQ (Figures 1 and 3GoGo). Another polar residue, Asp32, is located near TTQ with its amide N positioned 3.0 Å from the C7 oxygen of TTQ. Its side chain points away from the active site but makes hydrogen bonds with a water network that interacts with active-site residues. Each of these four residues is conserved in all MADHs that have been sequenced (Figure 2Go). Another residue, Phe55 of the {alpha} subunit, is also located at the opening of the active site. Previous site-directed mutagenesis studies have shown that this residue plays a role in dictating the substrate specificity of MADH (Zhu et al., 2000aGo).



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Fig. 3. Stereo diagram of the active site cavity of MADH from P.denitrificans (Chen et al., 1998Go) (Protein Data Bank entry 2BBK). Main and side chain atoms of three peptide segments, Ile31–Asp32–Gly33–Asn34, Asn104–Asp105–Ile106–Ile107–Trp108 and Tyr119–His120–Cys121–Thr122, plus Asp76 and the quinolated ring of TTQ that was derived from Trp57 (Trq57) are shown. The atoms are colored by atom type with carbon black, oxygen red, nitrogen blue and sulfur yellow. The view into the cavity is from the point of view of the side chain of Phe55 of the {alpha} subunit (not shown) which closes the cavity and appears to act as a gate for allowing access to the active site (Zhu et al., 2000aGo). Hydrogen bonds from the oxygens of Tyr119 OH, Thr122 OG1, Asp76 OD1 and OD2, Asp32 OD1 and O6 and O7 of TTQ are shown as dotted lines. The short contact distance (2.6 Å) between Asp32 OD1 and Asn104 O suggests that Asp32 may be protonated. This diagram was made using MOLSCRIPT and RASTER-3D.

 
The biosynthesis of MADH and TTQ is a complex process. It has been demonstrated that four gene products, in addition to the structural genes for the MADH subunits, are required for the biosynthesis of MADH (van der Palen et al., 1995Go; Graichen et al., 1999Go). An issue which has not previously been addressed is whether any of the conserved amino acid residues in the enzyme active site also play a role in the biosynthesis of MADH or the TTQ cofactor. Formation of the TTQ prosthetic group, which involves the covalent cross-linking of two distal tryptophan residues on the ß subunit, may contribute to the correct folding and stability of MADH. Conversely, correct folding of the protein and formation of intrasubunit disulfide bonds may be a prerequisite for the formation of TTQ. Some sort of linkage almost certainly exists between protein folding, intrasubunit disulfide bond formation, TTQ formation and protein stability.

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 3Go). 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

Native MADH was purified from P.denitrificans as described previously (Davidson, 1990Go). Site-directed mutants of MADH were expressed in Rhodobacter sphaeroides (Graichen et al., 1999Go) and purified as described previously (Zhu et al., 2000bGo). 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 IGo.


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Table I. Bacterial strains and plasmids
 
Construction of mutant MADH

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., 2000bGo). The primers used to create the desired mutations are shown in Table IIGo. 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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Table II. Primers used for site-directed mutagenesis
 
Kinetic analysis of MADH

The steady-state kinetic assay of MADH with phenazine ethosulfate as an electron acceptor was performed as described previously (Davidson, 1990Go). 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

(1)
where v is the measured initial reaction rate, E is the concentration of MADH, [S] is the concentration of methylamine, kcat is the turnover number and Km is the Michaelis–Menten constant.

Electrophoretic techniques

Whole cell extracts of R.sphaeroides expressing different site-directed mutants of MADH were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). 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, 1990Go). These extracts were subjected to non-denaturing SDS–PAGE 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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 Materials and methods
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Effects of site-directed mutations on the levels of detectable MADH activity

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 IIGo. 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 SDS–PAGE 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 4Go. 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 4Go, 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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Fig. 4. Activity staining of extracts of R.sphaeroides cells expressing site-directed mutants of MADH. Periplasmic extracts of cells were separated on a 12.5% non-denaturing polyacrylamide gels. The gel on the left was stained for total protein. The gel on the right was stained for MADH activity as described under Materials and methods. The lanes labeled MADH contained the purified protein. A122T* and F119Y* are back-mutations in which the T122A and Y119F mutants were converted back to wild-type by site-directed mutagenesis.

 
Extracts of cells expressing D76N and D32N MADH mutants were also analyzed for MADH activity. Activity was not readily detected in crude extracts. Activity staining of gels after non-denaturing PAGE reveled no detectable activity in extracts of cells expressing D76N MADH and weak but detectable activity in extracts of cells expressing D32N MADH. The results of the analysis of the extracts of cells expressing the different MADH mutants are summarized in Table IIIGo.


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Table III. Effects of site-directed mutations
 
Effects of site-directed mutations on the levels of detectable MADH ß subunit

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 5Go, Table IIIGo). 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 5Go. 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., 2000bGo), were included as a positive control. As seen in Figure 5Go, 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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Fig. 5. Western blot of extracts of R.sphaeroides cells expressing site-directed mutants of MADH. Whole cell extracts were prepared by sonication and were separated by SDS–PAGE. The gel on the left was stained for total protein. The Western blot on the right is of an identical gel which was probed using antibody raised against the MADH ß subunit. The lanes labeled MADH contained extracts from R.sphaeroides cells which expressed the wild-type recombinant MADH. F119Y* is a back-mutation in which the Y119F mutant was converted back to wild-type by site-directed mutagenesis.

 
Extracts of cells expressing D76N and D32N MADH mutants were also analyzed for the presence of the MADH ß subunit by Western blotting. A positive reaction, barely above our levels of detection, was observed with extracts of cells expressing D32N MADH. For the D76N mutant either no MADH or levels below our limit of detection were present in cell extracts. The results of the analysis of the extracts of cells expressing the different MADH mutants are summarized in Table IIIGo.

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 5GoGo 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 4Go). 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.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inspection of the crystal structure of MADH suggests that Asp76, Thr122 and Tyr119 participate in catalysis. The side chain oxygens of these residues are situated in the enzyme active site in close proximity to the reactive C6 carbonyl of TTQ. It is also apparent from the structure that Thr122 and Tyr119 play an important role in maintaining the proper folding of the protein. The ß subunits of MADH are mainly composed of two ß-sheet subdomains. These two subdomains are joined by an irregular coil containing two 3/10 helical turns, the second of which (residues 113–116) is close to the active site. The crystal structure of P.denitrificans MADH reveals that a strong hydrogen bond (i.e. a separation of 2.7 Å) exists between the side chains of Thr122 and Tyr119 (Figure 3Go). One effect of this hydrogen-bonding interaction is to pull the main chain atoms of Tyr119 closer to those of Thr122. As a consequence of the relative movement between them, a significant turn of the protein backbone of ~90° is observed. Moreover, the extended chain of the ß-strand beyond Tyr119 contains two ß-bulges and one observes a 180° twist of the protein backbone about its long axis (Figure 6Go). Maintenance of the overall secondary structure of course requires several bends and turns as well as several disulfide bridges. However, the 90° turn at residues 119–122 is part of the active site fold and the strong hydrogen bond between Thr122 and Tyr119 must help to stabilize it. The results presented in this paper support the notion that this specific hydrogen bond is an important factor for stabilizing the structure of the MADH ß subunit.



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Fig. 6. Ribbon diagram of the ß subunit of MADH from P.denitrificans. The ribbon color ramps red at the N-terminus to azure at the C-terminus. The positions of the mutated residues Asp32, Asp76, Tyr119 and Thr122 are shown. Also shown are the six disulfide bridges (labeled SS) and the cross-linked TTQ cofactor that help hold the structure together.

 
While removal of the hydroxyl moiety of either Thr122 or Tyr119 has devastating effects on MADH biosynthesis or stability, or both, there are interesting differences in the results obtained for site-directed mutants of Thr122 relative to those of Tyr119. For the T122A mutant, absolutely no detectable protein or activity was observed in cell extracts. For the Y119F mutant, active protein was detected, albeit at reduced levels relative to the wild-type. This raises the possibility that the hydroxyl moiety of Thr122 may play other significant roles in the biosynthesis or stability of the MADH ß subunit, in addition to its participation in the strong hydrogen bond with Tyr119. Thr122 is positioned such that it may potentially form two other hydrogen bonds in the enzyme active site. The side chain oxygen is ~3 Å from a side chain oxygen of Asp76 and ~3 Å from the O6 of TTQ. The oxygen of Asp76 is also ~3 Å from the O6 of TTQ. Our results are consistent with critical roles for Asp76 and Thr122 in catalysis. These results further show that the reactive oxygens on Thr122 and Asp76 are critical for MADH stability or biosynthesis or both, since no protein is detected after introduction of the T122A and D76N mutations. In the light of this, the hydrogen bonding interactions of Thr122 and Asp76 with the O6 of TTQ are particularly interesting, since the carbonyl oxygens of TTQ must be incorporated into Trp57 post-translationally. It is known that the biosynthesis of TTQ is not an autocatalytic process. Four genes in addition to those that encode the MADH structural genes are required for the complete biosynthesis of MADH (van der Palen et al., 1995Go; Graichen et al., 1999Go). This does not, however, preclude the possibility that amino acids on the ß subunit may also be involved in the biosynthetic process.

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, 1994Go; Matsuzaki et al., 1994Go). 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., 2000Go). 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., 1991Go; 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., 1998Go). 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., 1998Go). 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 {alpha} subunits of MADH. Consistent with this notion, it is noteworthy that while the previously described F55A mutation on the {alpha} 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, 1999Go).

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., 2000Go). 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., 1999Go). 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., 1990Go). 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., 1998Go). 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., 1998Go; Zhu and Davidson, 1999Go). Some evidence has previously been provided for roles of Tyr119 in determining the substrate specificity of MADH (Zhu et al., 2000aGo) and in monovalent cation binding (Labesse et al., 1998Go). 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, 1997Go). 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., 1994Go) 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.


    Notes
 
3 To whom correspondence should be addressed. E-mail: vdavidson{at}biochem.umsmed.edu Back


    Acknowledgments
 
This work was supported by NIH Grant GM-41574 (V.L.D.) and NSF Grant MCB-9728885 (F.S.M.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received March 12, 2001; revised June 15, 2001; accepted June 23, 2001.





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