Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase

We employed a highly specific photoaffinity labeling procedure, using (14)C-labeled S-adenosyl-l-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-(14)C]AdoMet or [carboxyl-(14)C]AdoMet yielded the sequence H(2)N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu(10654) and Pro(10655) as the major sites of derivatization. [carboxyl-(14)C]AdoMet in addition labeled Tyr(10650). Chymotryptic digestion generated the radiolabeled peptide H(2)N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu(2128) and Pro(2129), which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.     

Molecular dissection of the S-adenosylmethionine-binding site of phosphatidylethanolamine N-methyltransferase

Phosphatidylethanolamine N-methyltransferase (PEMT) is a quatrotopic membrane protein that catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine through three sequential methylation reactions. Analysis of mice lacking a functional PEMT gene revealed a severe reduction in plasma homocysteine levels. Homocysteine is generated by the hydrolysis of S-adenosylhomocysteine, which is also a product of the PEMT reaction. To gain insight into the PEMT transmethylation reaction and the mechanism by which PEMT regulates homocysteine levels, we sought to define residues that are required for binding of the methyl group donor, S-adenosylmethionine (AdoMet). Bioinformatic analysis of the predicted amino acid sequence of human PEMT identified two putative AdoMet-binding motifs (98GXG100 and 180EE181). Site-directed mutagenesis experiments demonstrated the requirement for the conserved motifs in PEMT specific activity. Analysis of the AdoMet binding ability of mutant recombinant PEMT derivatives established that residues Gly100 and Glu180 are essential for binding of the AdoMet moiety. A significantly elevated KD with respect to AdoMet is observed following conservative mutagenesis of residues Gly98 (400 pmol) and Glu181 (666.7 pmol), relative to the unmodified enzyme (303.1 pmol), suggesting that these residues also participate in AdoMet binding. A model positions two separate AdoMet-binding motifs of PEMT in close proximity at the external leaflet of the endoplasmic reticulum membrane.     

Correlating amino acid conservation with function in tyrosyl-tRNA synthetase

Sequence comparisons have been combined with mutational and kinetic analyses to elucidate how the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase evolved. Catalysis of tRNA(Tyr) aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine substrate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). Previous investigations indicate that the class I conserved KMSKS motif is involved in only the first step of the reaction (i.e. tyrosine activation). Here, we demonstrate that the class I conserved HIGH motif also is involved only in the tyrosine activation step. In contrast, one amino acid that is conserved in a subset of the class I aminoacyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 and Arg86, stabilize the transition states for both steps of the tRNA aminoacylation reaction. These results imply that stabilization of the transition state for the first step of the reaction by the class I aminoacyl-tRNA synthetases preceded stabilization of the transition state for the second step of the reaction. This is consistent with the hypothesis that the ability of aminoacyl-tRNA synthetases to catalyze the activation of amino acids with ATP preceded their ability to catalyze attachment of the amino acid to the 3' end of tRNA. We propose that the primordial aminoacyl-tRNA synthetases replaced a ribozyme whose function was to promote the reaction of amino acids and other small molecules with ATP.     

Comparative studies on S-adenosyl-l-methionine binding sites of protein N-methyltransferases,using 8-azido-S-adenosyl-l-methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Comparative studies on S-adenosyl-l -methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l -methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.     

Mutational analysis of conserved residues in HhaI DNA methyltransferase

HhaI DNA methyltransferase belongs to the C5-cytosine methyltransferase family, which is characterized by the presence of a set of highly conserved amino acids and motifs present in an invariant order. HhaI DNA methyltransferase has been subjected to a lot of biochemical and crystallographic studies. A number of issues, especially the role of the conserved amino acids in the methyltransferase activity, have not been addressed. Using sequence comparison and structural data, a structure-guided mutagenesis approach was undertaken, to assess the role of conserved amino acids in catalysis. Site-directed mutagenesis was performed on amino acids involved in cofactor S-adenosyl-L-methionine (AdoMet) binding (Phe18, Trp41, Asp60 and Leu100). Characterization of these mutants, by in vitro /in vivo restriction assays and DNA/AdoMet binding studies, indicated that most of the residues present in the AdoMet-binding pocket were not absolutely essential. This study implies plasticity in the recognition of cofactor by HhaI DNA methyltransferase.     

Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase.

Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies. Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast. In addition, polyclonal anti-E. coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes. Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity. Our results suggest that these conserved regions may be important in enzyme activity.     

Structure-function analysis of the human sialyltransferase ST3Gal I: role of n-glycosylation and a novel conserved sialylmotif

All eukaryotic sialyltransferases have in common the presence in their catalytic domain of several conserved peptide regions (sialylmotifs L, S, and VS). Functional analysis of sialylmotifs L and S previously demonstrated their involvement in the binding of donor and acceptor substrates. The region comprised between the sialylmotifs S and VS contains a stretch of four highly conserved residues, with the following consensus sequence (H/y)Y(Y/F/W/h)(E/D/q/g). (Capital letters and lowercase letters indicate a strong or low occurrence of the amino acid, respectively.) The functional importance of these residues and of the conserved residues of motif VS (HX(4)E) was assessed using as a template the human ST3Gal I. Mutational analysis showed that residues His(299) and Tyr(300) of the new motif, and His(316) of the VS motif, are essential for activity since their substitution by alanine yielded inactive enzymes. Our results suggest that the invariant Tyr residue (Tyr(300)) plays an important conformational role mainly attributable to the aromatic ring. In contrast, the mutants W301F, E302Q, and E321Q retained significant enzyme activity (25-80% of the wild type). Kinetic analyses and CDP binding assays showed that none of the mutants tested had any significant effect in nucleotide donor binding. Instead the mutant proteins were affected in their binding to the acceptor and/or demonstrated lower catalytic efficiency. Although the human ST3Gal I has four N-glycan attachment sites in its catalytic domain that are potentially glycosylated, none of them was shown to be necessary for enzyme activity. However, N-glycosylation appears to contribute to the proper folding and trafficking of the enzyme.     

Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Class I aminoacyl-tRNA synthetases (aaRSs) use a Rossmann-fold domain to catalyze the synthesis of aminoacyl-tRNAs required for decoding genetic information. While the Rossmann-fold domain is conserved in evolution, the acceptor stem near the aminoacylation site varies among tRNA substrates, raising the question of how the conserved protein fold adapts to RNA sequence variations. Of interest is the existence of an unpaired C-A mismatch at the 1-72 position unique to bacterial initiator tRNA(fMet) and absent from elongator tRNAs. Here we show that the class I methionyl-tRNA synthetase (MetRS) of Escherichia coli and its close structural homolog cysteinyl-tRNA synthetase (CysRS) display distinct patterns of recognition of the 1-72 base pair. While the structural homology of the two enzymes in the Rossmann-fold domain is manifested in a common burst feature of aminoacylation kinetics, CysRS discriminates against unpaired 1-72, whereas MetRS lacks such discrimination. A structure-based alignment of the Rossmann fold identifies the insertion of an α-helical motif, specific to CysRS but absent from MetRS, which docks on 1-72 and may discriminate against mismatches. Indeed, substitutions of the CysRS helical motif abolish the discrimination against unpaired 1-72. Additional structural alignments reveal that with the exception of MetRS, class I tRNA synthetases contain a structural motif that docks on 1-72. This work demonstrates that by flexible insertion of a structural motif to dock on 1-72, the catalytic domain of class I tRNA synthetases can acquire structural plasticity to adapt to changes at the end of the tRNA acceptor stem.     

Role for a conserved structural motif in assembly of a class I aminoacyl-tRNA synthetase active site

The catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates. Structural comparisons identified a conserved motif bridging the catalytic and anticodon binding domains of class Ia and Ib enzymes. This stem contact fold (SCF) has been proposed to globally orient each enzyme's cognate tRNA by interacting with the inner corner of the L-shaped tRNA. Despite the structural similarity of the SCF among class Ia/Ib enzymes, the sequence conservation is low. We replaced amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase (GlnRS) motif or with alanine residues. Chimeric variants retained significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity. In contrast, chimeras were significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain. A highly conserved aspartic acid within the MetRS SCF is proposed to make an electrostatic interaction with an active site lysine; these residues were replaced with alanines or conservative substitutions. Both methionyl adenylate formation and methionine transfer were impaired, and activity was not significantly recovered by making the compensatory double substitution.     

Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N6-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding.

Comparison of the deduced amino acid sequences of DNA-[N6-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY [or closely related] motif. By site-directed mutagenesis of a cloned T4 dam gene, we have altered the first proline residue in this motif [located in conserved region IV of the T4 Dam-MTase] to alanine or threonine. The mutant enzymic forms, P172A and P172T, were overproduced and purified. Kinetic studies showed that compared to the wild-type [wt] the two mutant enzymic forms had: (i) an increased [5 and 20-fold, respectively] Km for substrate, S-adenosyl-methionine [AdoMet]; (ii) a slightly reduced [2 and 4-fold lower] kcat; (iii) a strongly reduced kcat/KmAdoMet [10 and 100-fold]; and (iv) almost the same Km for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced [4 and 9-fold lower] Ka for AdoMet. Taken together these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPPY-motif is important for AdoMet-binding, and that region IV contains or is part of an AdoMet-binding site.     

Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding

Comparison of the deduced amino acid sequences of DNA-[N⁶-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY-motif, or a variant of it. By site-directed mutagenesis of a cloned T4 dam gene, we have altered the first proline residue in this motif (located in conserved region IV of the T4 Dam-MTase) to alanine or threonine. The mutant enzymic forms, P172A and P172T, were overproduced and purified. Kinetic studies showed that compared to the wild-type (wt) the two mutant enzymic forms had: (i) an increased (6 and 23-fold, respectively) K_m for substrate, S-adenosyl-methionine (AdoMet) and an increased (6 and 23-fold) K_i for product, S-adenosyl-homocysteine (AdoHcy); (ii) a slightly reduced (1.5 and 3-fold lower) k_cat; (iii) a strongly reduced k_cat/K_m^AdoMet (10 and 80-fold); and (iv) the same K_m for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced (3 and 7-fold lower) K_a for AdoMet; all forms bound two molecules of AdoMet. Taken together these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPPY-motif is important for AdoMet-binding, and that region IV contains an AdoMet-binding site.     

Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence.

Although partial or complete three-dimensional structures are known for three Class I aminoacyl-tRNA synthetases, the amino acid-binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl-tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl-tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.-P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449-457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300-fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the "signature sequence" of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl-tRNA synthetases and, in the known structures, it is in the nucleotide-binding fold as part of a loop between the end of a beta-strand and the start of an alpha-helix. The phenotype of the mutant and the stability and affinity for methionine of the wild-type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C-terminus of the signature sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of engineered multifunctional peptide synthetases. Enzymatic characterization of surfactin synthetase domains in hybrid bimodular systems.

The combinatorial reorganization of distinct modules of multimodular peptide synthetases is of increasing interest for the generation of new peptides with optimized bioactive properties. Each module is at least composed of enzymatic domains responsible for the adenylation, thioester formation, and condensation of an amino acid residue of the final peptide product. We analyzed various possible fusion sites for the recombination of peptide synthetases and evaluated the impact of different recombination strategies on the amino acid adenylation and acyl-thioester formation activities of peptide synthetase modules. Hybrid bimodular peptide synthetases were generated by recombination of the corresponding reading frames encoding for L-glutamic acid- and L-leucine-specific modules of surfactin synthetase SrfA-A at presumed inner- and intradomainic regions. We demonstrate that fusions at a previously postulated hinge region, dividing the amino acid adenylating domains of peptide synthetase modules into two subdomains, and at the highly conserved 4'-phosphopantetheine binding motif in acyl-thioester forming domains resulted in enzymatically active hybrid domains. By contrast, most manipulations in condensation domains like deletions, the complete exchange or the construction of chimeric domains considerably reduced or completely abolished the amino acid adenylation and thioester formation activity of the hybrid module.     

The tRNA-dependent activation of arginine by arginyl-tRNA synthetase requires inter-domain communication

The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized. A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels. A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line. The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5. The corresponding mutant enzymes were expressed in yeast and purified. The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP(i) exchange and tRNA aminoacylation reactions. This mutation is located underneath the floor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the alpha-helix bundle C-terminal domain distinctive of class 1a synthetases. For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates. However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo. In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic efficiency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme. In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the floor of the active site.     

The synthesis of the two S-adenosyl-methionine synthetases is differently regulated in Saccharomyces cerevisiae

Summary S-adenosyl-l-methionine (AdoMet) is synthesized by transfer of the adenosyl moiety of ATP to the sulfur atom of methionine. This reaction is catalysed by AdoMet synthetase. In all eukaryotic organisms studied so far, multiple forms of AdoMet synthetases have been reported and from their recent study, it appears that AdoMet synthetase is an exceptionally well conserved enzyme through evolution. In Saccharomyces cerevisiae, we have demonstrated the existence of two AdoMet synthetases encoded by genes SAM1 and SAM2. Yeast, which is able to concentrate exogenously added AdoMet, is thus a particularly useful biological system to understand the role and the physiological significance of the preservation of two almost identical AdoMet synthetases. The analysis of the expression of the two SAM genes in different genetic backgrounds during growth under different conditions shows that the expression of SAM1 and SAM2 is regulated differently. The regulation of SAM1 expression is identical to that of other genes implicated in AdoMet metabolism, where as SAM2 shows a specific pattern of regulation. A careful analysis of the expression of the two genes and of the variations in the methionine and AdoMet intracellular pools during the growth of different strains lead us to postulate the existence of two different AdoMet pools, each one suppplied by a different AdoMet synthetase but in equilibrium with each other. This could be a means of storing AdoMet whenever this metabolite is overproduced, thus avoiding the degradation of a metabolite the synthesis of which is energetically expensive.     

A relationship between asparagine synthetase A and aspartyl tRNA synthetase.

A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase.     

Mutational analysis of the Trypanosoma vivax alternative oxidase: the E(X)6Y motif is conserved in both mitochondrial alternative oxidase and plastid terminal oxidase and is indispensable for enzyme activity

Based on amino acid sequence similarity and the ability to catalyze the four-electron reduction of oxygen to water using a quinol substrate, mitochondrial alternative oxidase (AOX) and plastid terminal oxidase (PTOX) appear to be two closely related members of the membrane-bound diiron carboxylate group of proteins. In the current studies, we took advantage of the high activity of Trypanosoma vivax AOX (TvAOX) to examine the importance of the conserved Glu and the Tyr residues around the predicted third helix region of AOXs and PTOXs. We first compared the amino acid sequences of TvAOX with AOXs and PTOXs from various taxa and then performed alanine-scanning mutagenesis of TvAOX between amino acids Y(199) and Y(247). We found that the ubiquinol oxidase activity of TvAOX is completely lost in the E214A mutant, whereas mutants E215A and E216A retained more than 30% of the wild-type activity. Among the Tyr mutants, a complete loss of activity was also observed for the Y221A mutant, whereas the activities were equivalent to wild-type for the Y199A, Y212A, and Y247A mutants. Finally, residues Glu(214) and Tyr(221) were found to be strictly conserved among AOXs and PTOXs. Based on these findings, it appears that AOXs and PTOXs are a novel subclass of diiron carboxylate proteins that require the conserved motif E(X)(6)Y for enzyme activity.