Use of Indirect Site-directed Mutagenesis to Alter the Substrate Specificity of Methylamine Dehydrogenase
2002; Elsevier BV; Volume: 277; Issue: 6 Linguagem: Inglês
10.1074/jbc.m109270200
ISSN1083-351X
AutoresYongting Wang, Dapeng Sun, Victor L. Davidson,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoMethylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone-dependent enzyme that catalyzes the oxidative deamination of primary amines. Native MADH exhibits a strong preference for methylamine over longer carbon chain amines. Residue αPhe55 controls this substrate specificity. When αPhe55 is replaced with Ala, this preference is reversed with αF55A MADH preferring long-chain amines with at least seven carbons (Zhu, Z., Sun, D., and Davidson, V. L. (2000)Biochemistry 39, 11184–11186). To further modulate the substrate specificity of MADH, the side-chain of αPhe55was repositioned by site-directed mutagenesis of residue βIle107. This residue makes close contact with αPhe55 and restricts its movement. βI107V MADH exhibits a strong preference for propylamine, and βI107N MADH exhibits a preference for 1-aminopentane. Thus, it has been possible to create forms of MADH that exhibit a preference for amines with carbon chain lengths of one, three, five, or seven carbons. The ability to discriminate between amines of different chain length was essentially abolished by an αF55I mutation. Molecular modeling studies with the known crystal structure are described that provide an explanation for these results. These results provide an example of a design-based approach to protein engineering in which site-directed mutagenesis on one residue can be used to reposition another residue to specifically alter enzyme specificity. Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone-dependent enzyme that catalyzes the oxidative deamination of primary amines. Native MADH exhibits a strong preference for methylamine over longer carbon chain amines. Residue αPhe55 controls this substrate specificity. When αPhe55 is replaced with Ala, this preference is reversed with αF55A MADH preferring long-chain amines with at least seven carbons (Zhu, Z., Sun, D., and Davidson, V. L. (2000)Biochemistry 39, 11184–11186). To further modulate the substrate specificity of MADH, the side-chain of αPhe55was repositioned by site-directed mutagenesis of residue βIle107. This residue makes close contact with αPhe55 and restricts its movement. βI107V MADH exhibits a strong preference for propylamine, and βI107N MADH exhibits a preference for 1-aminopentane. Thus, it has been possible to create forms of MADH that exhibit a preference for amines with carbon chain lengths of one, three, five, or seven carbons. The ability to discriminate between amines of different chain length was essentially abolished by an αF55I mutation. Molecular modeling studies with the known crystal structure are described that provide an explanation for these results. These results provide an example of a design-based approach to protein engineering in which site-directed mutagenesis on one residue can be used to reposition another residue to specifically alter enzyme specificity. methylamine dehydrogenase tryptophan tryptophylquinone Methylamine dehydrogenase (MADH)1 (EC 1.4.99.3) fromParacoccus denitrificans is a soluble bacterial enzyme that catalyzes the oxidative deamination of methylamine to formaldehyde plus ammonia (1Davidson V.L. Adv. Protein Chem. 2001; 58: 95-140Crossref PubMed Scopus (93) Google Scholar, 2McIntire W.S. Wemmer D.E. Christoserdov A.Y. Lindstrom M.E. Science. 1991; 252: 817-824Crossref PubMed Scopus (313) Google Scholar). It possesses an α2β2structure and subunit molecular weights of 46,700 and 15,500 (3Chen L. Doi M. Durley R.C. Chistoserdov A.Y. Lidstrom M.E. Davidson V.L. Mathews F.S. J. Mol. Biol. 1998; 276: 131-149Crossref PubMed Scopus (99) Google Scholar). Each smaller β subunit possesses a covalently bound tryptophan tryptophylquinone (TTQ) prosthetic group, which is formed by post-translational modifications of Trp57 and Trp108 of the β subunit (2McIntire W.S. Wemmer D.E. Christoserdov A.Y. Lindstrom M.E. Science. 1991; 252: 817-824Crossref PubMed Scopus (313) Google Scholar). During catalysis the substrate amine forms a covalent bond with the C-6 carbonyl of TTQ (4Huizinga E.G. van Zanten B.A. Duine J.A. Jongejan J.A. Huitema F. Wilson K.S. Hol W.G. Biochemistry. 1992; 31: 9789-9795Crossref PubMed Scopus (34) Google Scholar, 5Labesse G. Ferrari D. Chen Z.W. Rossi G.L. Kuusk V. McIntire W.S. Mathews F.S. J. Biol. Chem. 1998; 273: 25703-25712Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) in the enzyme active site. This active site is connected to the enzyme surface by a short substrate channel that comprises residues from both subunits. The side-chains of αPhe55 and βTyr119 form a “gate” where the substrate channel opens into the active site (3Chen L. Doi M. Durley R.C. Chistoserdov A.Y. Lidstrom M.E. Davidson V.L. Mathews F.S. J. Mol. Biol. 1998; 276: 131-149Crossref PubMed Scopus (99) Google Scholar, 6Zhu Z. Sun D. Davidson V.L. Biochemistry. 2000; 39: 11184-11186Crossref PubMed Scopus (24) Google Scholar). The phenyl group of αPhe55 serves two functions in determining substrate specificity. It interacts with the methyl group of methylamine to help orient the amino group of the substrate for nucleophilic attack of TTQ, and it excludes long-chain amines from the active site (Fig.1). Site-directed mutagenesis was previously used to create αF55A MADH, which exhibited a dramatically altered substrate specificity in which long-chain amines are preferred to short-chain amines (6Zhu Z. Sun D. Davidson V.L. Biochemistry. 2000; 39: 11184-11186Crossref PubMed Scopus (24) Google Scholar).In this paper we further examine the extent to which the nature and position of the side-chain of residue α55 can determine the substrate specificity of MADH. In addition to directly mutating residue αPhe55, site-directed mutagenesis was used to alter the position of this residue. To reposition the phenyl side-chain of αPhe55, mutations were made of residue Ile107 of the β subunit. This residue is located adjacent to αPhe55 at the interface of the α and β subunits. From the crystal structure, it appears that the side-chain of βIle107 restricts the movement of the side-chain of αPhe55. We show that relatively conservative mutations of βIle107 significantly alter the substrate specificity of MADH. When βIle107 is converted to valine, the enzyme exhibits a strong preference for propylamine. When βIle107 is converted to asparagine, the preferred substrate is the five-carbon amylamine (1-aminopentane). Consistent with the importance of the position of the phenyl ring of αPhe55 in determining substrate specificity, conversion of αPhe55 to isoleucine yields an enzyme that exhibits no clear substrate specificity and shows relatively poor activity with amines of any carbon chain length. Thus, it has been possible to use site-directed mutagenesis to create forms of MADH that exhibit preference for amines with carbon chain lengths of one, three, five, or seven carbons; or which exhibit no preference at all. Molecular modeling studies with the known crystal structure are used to provide an explanation for these results.EXPERIMENTAL PROCEDURESNative MADH was purified from P. denitrificans as described previously (7Davidson V.L. Methods Enzymol. 1990; 188: 241-246Crossref PubMed Scopus (79) Google Scholar). The βI107V, βI107N, and αF55I mutants of MADH were heterologously expressed in Rhodobacter sphaeroides (8Graichen M.E. Jones L.H. Sharma B.V. van Spanning R.J. Hosler J.P. Davidson V.L. J. Bacteriol. 1999; 181: 4216-4222Crossref PubMed Google Scholar) and purified as described previously for the αF55A MADH mutant (9Zhu Z. Jones L.H. Graichen M.E. Davidson V.L. Biochemistry. 2000; 39: 8830-8836Crossref PubMed Scopus (17) Google Scholar). All reagents were purchased from Sigma or Aldrich and used without further purification.Site-directed mutagenesis was performed on double-stranded pMEG976 (8Graichen M.E. Jones L.H. Sharma B.V. van Spanning R.J. Hosler J.P. Davidson V.L. J. Bacteriol. 1999; 181: 4216-4222Crossref PubMed Google Scholar) using the QuickChange Site-directed Mutagenesis Kit (Stratagene) and two mutagenic primers following a previously described procedure (8Graichen M.E. Jones L.H. Sharma B.V. van Spanning R.J. Hosler J.P. Davidson V.L. J. Bacteriol. 1999; 181: 4216-4222Crossref PubMed Google Scholar). In this construct, the gene that encodes the MADH β subunit possesses a polyhistidine tag at its C terminus to facilitate purification of the expressed protein. The primers used to create the site-directed mutations were: βI107V, 5′-CAACGACATCGTCTGGTGCTTCGGCGCCGAGG-3′; βI107N, 5′-CAACGACATCAACTGGTGCTTCGGCGCCGAGG-3′; αF55I, 5′-GTCAACGACCCGGCGCATATCGCCGCGGTCACCCAGCAATTCG-3′. For each, the complementary sequence was used as the second primer for the mutagenesis. The underlined bases are those that were changed to create the desired mutation. The mutations were confirmed by sequencing 70 base pairs around the mutated site.Steady-state kinetic assays (7Davidson V.L. Methods Enzymol. 1990; 188: 241-246Crossref PubMed Scopus (79) Google Scholar) were performed in 10 mmpotassium phosphate, pH 7.5, at 30 °C. The assay mixture contained 16 nm MADH, varied concentrations of substrates, 4.8 mm phenazine ethosulfate, and 170 μm2,6-dichlorophenolindophenol. The reaction was monitored at 600 nm to determine the rate of reduction of 2,6-dichlorophenolindophenol. Data were fit to equation 1,v/E=kcat[S]/(Km+[S])Equation 1 where v is the measured initial rate, E is the MADH concentration, [S] is the substrate concentration,kcat is the turnover number, andKm is the Michaelis constant.Molecular modeling was performed using the QUANTA and CHARMm (Molecular Simulations) computer programs run on a Silicon Graphics O2 computer. The crystal structure of MADH that was used is Protein Data Bank entry2BBK (3Chen L. Doi M. Durley R.C. Chistoserdov A.Y. Lidstrom M.E. Davidson V.L. Mathews F.S. J. Mol. Biol. 1998; 276: 131-149Crossref PubMed Scopus (99) Google Scholar).RESULTS AND DISCUSSIONNative MADH exhibits a strong preference for methylamine, andKm increases with increasing length of the carbon chain (10Davidson V.L. Biochem. J. 1989; 261: 107-111Crossref PubMed Scopus (43) Google Scholar). For αF55A MADH the preference is reversed with the enzyme preferring long-chain amines, and Km increases with decreasing length of the carbon chain (6Zhu Z. Sun D. Davidson V.L. Biochemistry. 2000; 39: 11184-11186Crossref PubMed Scopus (24) Google Scholar). Steady-state kinetic analysis with a variety of amines as substrates showed that the substrate specificities of βI107V, βI107N, and αF55I MADH were each different from that of either the native MADH or αF55A MADH (Table I). Monoamines longer than amylamine (1-aminopentane) are not soluble enough in aqueous solution to achieve concentrations necessary for the kinetic studies. Primary 1,N-diamines, which are more soluble than their corresponding monoamines, were tested as substrates to examine the effect of increasing the carbon chain length beyond five. The βI107V MADH exhibits a relatively strong preference for propylamine withKm increasing as the carbon chain length is either increased or decreased. The Km value for propylamine is approximately the same as the Km value that native MADH exhibits for methylamine. The βI107N MADH exhibits a preference for 1-aminopentane with Km increasing as the carbon chain length is either increased or decreased. TheKm value for 1-aminopentane is also approximately the same as the Km value that native MADH exhibits for methylamine. The same patterns of substrate preference for these enzymes are indicated whenkcat/Km values are compared (Fig. 2) as when examining theKm values.Table ISubstrate specificities of native and mutant MADHsSubstrateNative MADHβI107V MADHβI107N MADHαF55A MADHαF55I MADHKmkcatKmkcatKmkcatKmkcatKmkcatμms−1μms−1μms−1μms−1μms−1Methylamine9 ± 130 ± 269 ± 620 ± 1250 ± 2234 ± 114900 ± 110077 ± 260 ± 82.0 ± 0.1Ethylamine19 ± 124 ± 1340 ± 596.2 ± 0.4840 ± 804.5 ± 0.29200 ± 130023 ± 1360 ± 4015 ± 1Propylamine36 ± 227 ± 16 ± 13.1 ± 0.18 ± 14.1 ± 0.21300 ± 15024 ± 1200 ± 125.2 ± 0.1Butylamine870 ± 5922 ± 188 ± 914 ± 17 ± 14.2 ± 0.1240 ± 2834 ± 1370 ± 405.4 ± 0.11-Aminopentane2500 ± 29017 ± 1130 ± 253.4 ± 0.24 ± 14.2 ± 0.247 ± 520 ± 1250 ± 215.4 ± 0.81,6-Diaminohexane720 ± 8317 ± 3170 ± 1026 ± 131 ± 316 ± 3121 ± 7343 ± 699 ± 63.4 ± 0.11,7-Diaminoheptane380 ± 4627 ± 1290 ± 4019 ± 268 ± 715 ± 17 ± 132 ± 157 ± 43.4 ± 0.1 Open table in a new tab Figure 2Effects of site-directed mutagenesis onkcat/Km values for different carbon chain length amines. Data are taken from Table I. Values are normalized to thekcat/Km value that each MADH exhibits for its preferred substrate. The actual maximum values are: MADH (▪), 3.3 × 106m−1s−1 for methylamine; βI107V MADH (▴), 5.2 × 105m−1 s−1 for propylamine; βI107N MADH (▾), 1.1 × 106m−1 s−1 for 1-aminopentane; αF55A MADH (♦), 4.6 × 106m−1 s−1 for 1,7-diaminoheptane.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In contrast to the other site-directed mutations, conversion of αPhe55 to Ile yields an αF55I MADH, which shows very little ability to discriminate between amines with different carbon chain lengths, and for each amine tested the Kmvalue is at least 100-fold greater than that of theKm value for the preferred substrate of each of the other MADHs. The molecular basis for these dramatic changes in substrate specificity that are caused by relatively conservative single site-directed mutations may be inferred from analysis of the crystal structure of MADH.Previous studies have suggested that the position of αPhe55 is important for stabilizing interactions with the methyl group of methylamine and that steric hindrance restricted entry of amines with carbon chain lengths of three or more (6Zhu Z. Sun D. Davidson V.L. Biochemistry. 2000; 39: 11184-11186Crossref PubMed Scopus (24) Google Scholar). This can be seen in Fig. 1 in which a five-carbon amine has been modeled into the active site of MADH. The crystal structure of native MADH further shows that the δ1-methyl group of βIle107 points toward the center of phenyl ring of αPhe55 (Fig.3). The distance between the δ1C of βIle107 and the δ1C of αPhe55 is ∼4.0 Å. Thus, βIle107 restricts the rotation of the phenyl group of αPhe55. This methyl group is removed by the βI107V mutation. Thus, substitution of valine for isoleucine will allow limited rotation of the phenyl ring of αPhe55. One can see in Fig. 4 that a small rotation of the phenyl ring about its bond with its β-carbon could reposition this residue such that the stabilizing interaction for the C-1 carbon of the substrate amine is lost, and stabilizing van der Waals' interactions with the C-3 carbon of the substrate may occur. The specificity would decrease as the carbon chain length increases beyond three because there will be an overlap of van der Waals' radii between the longer chain substrates and βTyr119. This accounts for the strong preference of βI107V MADH for propylamine over longer and shorter amines.Figure 3Relative positions of βIle107 with αPhe55 and βTyr119. These residues are drawn as space-filling models with heteroatoms indicated by darker shading. The δ1 and γ2 carbons of βIle107, which appear to restrict the movements of the side-chains of αPhe55 and βTyr119, respectively, are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Stabilization of propylamine in the active site after repositioning of αPhe55. Propylamine has been modeled into the active site of MADH. TTQ, αPhe55, βTyr119, and the amine are drawn as space-filling models with heteroatoms indicated by darker shading. The phenyl ring of αPhe55 has been rotated about the bond with its β-carbon so that it now makes van der Waals' interactions with the C-3 of propylamine, which helps to orient it for reaction with TTQ.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The δ1-methyl group of βIle107 is also removed by the βI107N mutation; however, βI107N MADH prefers amines with carbon chain lengths of five rather than three. Inspection of the crystal structure reveals that the γ2-methyl group of βIle107points toward the β-methyl group of βTyr119 (Fig. 3) and may help to orient the position of the phenol ring of βTyr119. The distance between the γ2C of βIle107 and the βC of βTyr119 is ∼4.2 Å. In βI107N MADH, this interaction is also eliminated. Therefore, the position of the phenol ring could be more flexible with less restricted rotation around the γC–βC and βC–αC bonds of βTyr119. At the same time, the amide group of asparagine has a smaller van der Waals' radius than the corresponding γ1- and δ1-methyl groups of Ile, which also gives αPhe55 more freedom of rotation. These two factors together make a more flexible gate to the active site in βI107N MADH than in native MADH. Thus, the substrate preference for βI107N MADH is also not as strict as was that of βI107V MADH. Although the five-carbon chain is preferred, propylamine and butylamine are not that much poorer substrates.Substitution of isoleucine for αPhe55 essentially eliminates the substrate specificity of MADH, converting it to an enzyme that is a relatively poor catalyst for the oxidative deamination of primary amines without regard for their carbon chain length. The most likely explanation for this is that the Ile side-chain is free to rotate about several carbon–carbon bonds and cannot be placed into a rigid conformation like that of αPhe55 in the native enzyme. As such, there is less restriction of long-chain amines but also less specific stabilization of short-chain amines.It is difficult to analyze these data in terms of a quantitative comparative binding analysis because these are steady-stateKm values and not true association constants. Some qualitative comments, however, are in order. It is noteworthy that theKm values for the most preferred substrate for the native, βI107V, βI107N, and αF55A MADHs are approximately the same. For the reaction of native MADH with methylamine, theKd value of 13 μm that was obtained from transient kinetic studies (11Brooks H.B. Jones L.H. Davidson V.L. Biochemistry. 1993; 32: 2725-2729Crossref PubMed Scopus (87) Google Scholar) is similar to the steady-stateKm value (Table I). If we regard the relative values of ln(1/Km) as an indication of the relative values of ΔG°, this therefore suggests that the sum of the binding energies that stabilize the enzyme-substrate complex are approximately the same for each mutant with its preferred substrate. This means that these mutations need not have changed the mechanism or nature of the interactions that stabilize substrate binding. They have simply changed the position at which the stabilizing interactions occur on substrates of different carbon chain lengths. This is consistent with our earlier discussion.There are two main factors affecting substrate preference of MADH. One is the ability of αPhe55 and βTyr119 to stabilize the binding of the amine substrate and orient its amino group for nucleophilic attack of TTQ. The other function of αPhe55 and βTyr119 is to exclude long-chain amines from the active site due to steric hindrance. These facts have been demonstrated by site-directed mutagenesis and exploited to specifically alter the substrate preference of MADH. Single site-directed mutations of residues αPhe55 and βIle107 of MADH significantly change the substrate specificity of this enzyme. The results show that MADH can be changed from an enzyme with a strong preference for methylamine to enzymes that prefer amines with carbon chain lengths of three (βI107V), five (βI107N), or seven or more (αF55A). A single mutation was also used to render MADH unable to discriminate between amines with varying length carbon chains (αF55I).From a protein engineering perspective, it is desirable to develop simple approaches for altering substrate specificity of enzymes without causing major structural changes that could otherwise affect the function or stability of the enzyme, or both. There are relatively few examples of the use of site-directed mutagenesis to rationally redesign substrate specificity. In most cases, multiple changes are required. For example, the substrate preference of aspartate aminotransferase was altered using homology modeling to prefer tyrosine (12Onuffer J.J. Kirsch J.F. Protein Sci. 1995; 4: 1750-1757Crossref PubMed Scopus (71) Google Scholar) and using directed evolution to prefer branched-chain amino acids (13Yano T. Oue S. Kagamiyama H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5511-5515Crossref PubMed Scopus (171) Google Scholar). However, in each case it was necessary to change at least six amino acid residues to alter the substrate specificity. Trypsin has been converted to chymotrypsin by replacing two surface loops containing 13 amino acids (14Hedstrom L. Szilagyi L. Rutter W.J. Science. 1992; 255: 1249-1253Crossref PubMed Scopus (450) Google Scholar). It has been possible to alter substrate specificity by single amino acid substitutions. Examples of this include isocitrate dehydrogenase (15Doyle S.A. Fung S-Y.F. Koshland D.E. Biochemistry. 2000; 39: 14348-14355Crossref PubMed Scopus (21) Google Scholar) and glutathione reductase (16Stoll V.S. Simpson S.J. Krauth-Siegel L. Walsh C.T. Pai E.F. Biochemistry. 1997; 36: 6437-6447Crossref PubMed Scopus (46) Google Scholar). These results showed that interactions between neighboring residues in the active site may strongly influence each other. In the latter case, it was shown that an A34E mutation caused a significant movement of Arg347 due to steric hindrance, which in turn altered substrate specificity. We report here how it was possible to use a rational approach to redesign substrate specificity by using site-directed mutagenesis of a single amino acid residue to reposition another critical residue in the active site. The demonstration with MADH that the specificity of the enzyme may be modulated over a wide range of substrates, in a predictable manner, by single mutations suggests that such an approach may be applicable to other enzymes. Methylamine dehydrogenase (MADH)1 (EC 1.4.99.3) fromParacoccus denitrificans is a soluble bacterial enzyme that catalyzes the oxidative deamination of methylamine to formaldehyde plus ammonia (1Davidson V.L. Adv. Protein Chem. 2001; 58: 95-140Crossref PubMed Scopus (93) Google Scholar, 2McIntire W.S. Wemmer D.E. Christoserdov A.Y. Lindstrom M.E. Science. 1991; 252: 817-824Crossref PubMed Scopus (313) Google Scholar). It possesses an α2β2structure and subunit molecular weights of 46,700 and 15,500 (3Chen L. Doi M. Durley R.C. Chistoserdov A.Y. Lidstrom M.E. Davidson V.L. Mathews F.S. J. Mol. Biol. 1998; 276: 131-149Crossref PubMed Scopus (99) Google Scholar). Each smaller β subunit possesses a covalently bound tryptophan tryptophylquinone (TTQ) prosthetic group, which is formed by post-translational modifications of Trp57 and Trp108 of the β subunit (2McIntire W.S. Wemmer D.E. Christoserdov A.Y. Lindstrom M.E. Science. 1991; 252: 817-824Crossref PubMed Scopus (313) Google Scholar). During catalysis the substrate amine forms a covalent bond with the C-6 carbonyl of TTQ (4Huizinga E.G. van Zanten B.A. Duine J.A. Jongejan J.A. Huitema F. Wilson K.S. Hol W.G. Biochemistry. 1992; 31: 9789-9795Crossref PubMed Scopus (34) Google Scholar, 5Labesse G. Ferrari D. Chen Z.W. Rossi G.L. Kuusk V. McIntire W.S. Mathews F.S. J. Biol. Chem. 1998; 273: 25703-25712Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) in the enzyme active site. This active site is connected to the enzyme surface by a short substrate channel that comprises residues from both subunits. The side-chains of αPhe55 and βTyr119 form a “gate” where the substrate channel opens into the active site (3Chen L. Doi M. Durley R.C. Chistoserdov A.Y. Lidstrom M.E. Davidson V.L. Mathews F.S. J. Mol. Biol. 1998; 276: 131-149Crossref PubMed Scopus (99) Google Scholar, 6Zhu Z. Sun D. Davidson V.L. Biochemistry. 2000; 39: 11184-11186Crossref PubMed Scopus (24) Google Scholar). The phenyl group of αPhe55 serves two functions in determining substrate specificity. It interacts with the methyl group of methylamine to help orient the amino group of the substrate for nucleophilic attack of TTQ, and it excludes long-chain amines from the active site (Fig.1). Site-directed mutagenesis was previously used to create αF55A MADH, which exhibited a dramatically altered substrate specificity in which long-chain amines are preferred to short-chain amines (6Zhu Z. Sun D. Davidson V.L. Biochemistry. 2000; 39: 11184-11186Crossref PubMed Scopus (24) Google Scholar). In this paper we further examine the extent to which the nature and position of the side-chain of residue α55 can determine the substrate specificity of MADH. In addition to directly mutating residue αPhe55, site-directed mutagenesis was used to alter the position of this residue. To reposition the phenyl side-chain of αPhe55, mutations were made of residue Ile107 of the β subunit. This residue is located adjacent to αPhe55 at the interface of the α and β subunits. From the crystal structure, it appears that the side-chain of βIle107 restricts the movement of the side-chain of αPhe55. We show that relatively conservative mutations of βIle107 significantly alter the substrate specificity of MADH. When βIle107 is converted to valine, the enzyme exhibits a strong preference for propylamine. When βIle107 is converted to asparagine, the preferred substrate is the five-carbon amylamine (1-aminopentane). Consistent with the importance of the position of the phenyl ring of αPhe55 in determining substrate specificity, conversion of αPhe55 to isoleucine yields an enzyme that exhibits no clear substrate specificity and shows relatively poor activity with amines of any carbon chain length. Thus, it has been possible to use site-directed mutagenesis to create forms of MADH that exhibit preference for amines with carbon chain lengths of one, three, five, or seven carbons; or which exhibit no preference at all. Molecular modeling studies with the known crystal structure are used to provide an explanation for these results. EXPERIMENTAL PROCEDURESNative MADH was purified from P. denitrificans as described previously (7Davidson V.L. Methods Enzymol. 1990; 188: 241-246Crossref PubMed Scopus (79) Google Scholar). The βI107V, βI107N, and αF55I mutants of MADH were heterologously expressed in Rhodobacter sphaeroides (8Graichen M.E. Jones L.H. Sharma B.V. van Spanning R.J. Hosler J.P. Davidson V.L. J. Bacteriol. 1999; 181: 4216-4222Crossref PubMed Google Scholar) and purified as described previously for the αF55A MADH mutant (9Zhu Z. Jones L.H. Graichen M.E. Davidson V.L. Biochemistry. 2000; 39: 8830-8836Crossref PubMed Scopus (17) Google Scholar). All reagents were purchased from Sigma or Aldrich and used without further purification.Site-directed mutagenesis was performed on double-stranded pMEG976 (8Graichen M.E. Jones L.H. Sharma B.V. van Spanning R.J. Hosler J.P. Davidson V.L. J. Bacteriol. 1999; 181: 4216-4222Crossref PubMed Google Scholar) using the QuickChange Site-directed Mutagenesis Kit (Stratagene) and two mutagenic primers following a previously described procedure (8Graichen M.E. Jones L.H. Sharma B.V. van Spanning R.J. Hosler J.P. Davidson V.L. J. Bacteriol. 1999; 181: 4216-4222Crossref PubMed Google Scholar). In this construct, the gene that encodes the MADH β subunit possesses a polyhistidine tag at its C terminus to facilitate purification of the expressed protein. The primers used to create the site-directed mutations were: βI107V, 5′-CAACGACATCGTCTGGTGCTTCGGCGCCGAGG-3′; βI107N, 5′-CAACGACATCAACTGGTGCTTCGGCGCCGAGG-3′; αF55I, 5′-GTCAACGACCCGGCGCATATCGCCGCGGTCACCCAGCAATTCG-3′. For each, the complementary sequence was used as the second primer for the mutagenesis. The underlined bases are those that were changed to create the desired mutation. The mutations were confirmed by sequencing 70 base pairs around the mutated site.Steady-state kinetic assays (7Davidson V.L. Methods Enzymol. 1990; 188: 241-246Crossref PubMed Scopus (79) Google Scholar) were performed in 10 mmpotassium phosphate, pH 7.5, at 30 °C. The assay mixture contained 16 nm MADH, varied concentrations of substrates, 4.8 mm phenazine ethosulfate, and 170 μm2,6-dichlorophenolindophenol. The reaction was monitored at 600 nm to determine the rate of reduction of 2,6-dichlorophenolindophenol. Data were fit to equation 1,v/E=kcat[S]/(Km+[S])Equation 1 where v is the measured initial rate, E is the MADH concentration, [S] is the substrate concentration,kcat is the turnover number, andKm is the Michaelis constant.Molecular modeling was performed using the QUANTA and CHARMm (Molecular Simulations) computer programs run on a Silicon Graphics O2 computer. The crystal structure of MADH that was used is Protein Data Bank entry2BBK (3Chen L. Doi M. Durley R.C. Chistoserdov A.Y. Lidstrom M.E. Davidson V.L. Mathews F.S. J. Mol. Biol. 1998; 276: 131-149Crossref PubMed Scopus (99) Google Scholar). Native MADH was purified from P. denitrificans as described previously (7Davidson V.L. Methods Enzymol. 1990; 188: 241-246Crossref PubMed Scopus (79) Google Scholar). The βI107V, βI107N, and αF55I mutants of MADH were heterologously expressed in Rhodobacter sphaeroides (8Graichen M.E. Jones L.H. Sharma B.V. van Spanning R.J. Hosler J.P. Davidson V.L. J. Bacteriol. 1999; 181: 4216-4222Crossref P
Referência(s)