A threonine on the active site loop controls transition state formation in Escherichia coli respiratory complex II

In Escherichia coli, the complex II superfamily members succinate:ubiquinone oxidoreductase (SQR) and quinol:fumarate reductase (QFR) participate in aerobic and anaerobic respiration, respectively. Complex II enzymes catalyze succinate and fumarate interconversion at the interface of two domains of the soluble flavoprotein subunit, the FAD binding domain and the capping domain. An 11-amino acid loop in the capping domain (Thr-A234 to Thr-A244 in quinol:fumarate reductase) begins at the interdomain hinge and covers the active site. Amino acids of this loop interact with both the substrate and a proton shuttle, potentially coordinating substrate binding and the proton shuttle protonation state. To assess the loop's role in catalysis, two threonine residues were mutated to alanine: QFR Thr-A244 (act-T; Thr-A254 in SQR), which hydrogen-bonds to the substrate at the active site, and QFR Thr-A234 (hinge-T; Thr-A244 in SQR), which is located at the hinge and hydrogen-bonds the proton shuttle. Both mutations impair catalysis and decrease substrate binding. The crystal structure of the hinge-T mutation reveals a reorientation between the FAD-binding and capping domains that accompanies proton shuttle alteration. Taken together, hydrogen bonding from act-T to substrate may coordinate with interdomain motions to twist the double bond of fumarate and introduce the strain important for attaining the transition state.     

Structure of FAD-bound L-aspartate oxidase: insight into substrate specificity and catalysis

L-Aspartate oxidase (Laspo) catalyzes the conversion of L-Asp to iminoaspartate, the first step in the de novo biosynthesis of NAD(+). This bacterial pathway represents a potential drug target since it is absent in mammals. The Laspo R386L mutant was crystallized in the FAD-bound catalytically competent form and its three-dimensional structure determined at 2.5 A resolution in both the native state and in complex with succinate. Comparison of the R386L holoprotein with the wild-type apoenzyme [Mattevi, A., Tedeschi, G., Bacchella, L., Coda, A., Negri, A., and Ronchi, S. (1999) Structure 7, 745-756] reveals that cofactor incorporation leads to the ordering of two polypeptide segments (residues 44-53 and 104-141) and to a 27 degree rotation of the capping domain. This motion results in the formation of the active site cavity, located at the interface between the capping domain and the FAD-binding domain. The structure of the succinate complex indicates that the cavity surface is decorated by two clusters of H-bond donors that anchor the ligand carboxylates. Moreover, Glu121, which is strictly conserved among Laspo sequences, is positioned to interact with the L-Asp alpha-amino group. The architecture of the active site of the Laspo holoenzyme is remarkably similar to that of respiratory fumarate reductases, providing strong evidence for a common mechanism of catalysis in Laspo and flavoproteins of the succinate dehydrogenase/fumarate reductase family. This implies that Laspo is mechanistically distinct from other flavin-dependent amino acid oxidases, such as the prototypical D-amino acid oxidase.     

A third crystal form of Wolinella succinogenes quinol:fumarate reductase reveals domain closure at the site of fumarate reduction.

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone. Previously, the crystal structure of QFR from Wolinella succinogenes was determined based on two different crystal forms, and the site of fumarate binding in the flavoprotein subunit A of the enzyme was located between the FAD-binding domain and the capping domain [Lancaster, C.R.D., Kr?ger, A., Auer, M., & Michel, H. (1999) Nature 402, 377--385]. Here we describe the structure of W. succinogenes QFR based on a third crystal form and refined at 3.1 A resolution. Compared with the previous crystal forms, the capping domain is rotated in this structure by approximately 14 degrees relative to the FAD-binding domain. As a consequence, the topology of the dicarboxylate binding site is much more similar to those of membrane-bound and soluble fumarate reductase enzymes from other organisms than to that found in the previous crystal forms of W. succinogenes QFR. This and the effects of the replacement of Arg A301 by Glu or Lys by site-directed mutagenesis strongly support a common mechanism for fumarate reduction in this superfamily of enzymes.     

Crystal structures of the conserved tRNA-modifying enzyme GidA: implications for its interaction with MnmE and substrate

GidA is a flavin-adenine-dinucleotide (FAD)-binding protein that is conserved among bacteria and eucarya. Together with MnmE, it is involved in the addition of a carboxymethylaminomethyl group to the uridine base in the wobble position (nucleotide 34) of tRNAs that read split codon boxes. Here, we report the crystal structures of the GidA proteins from both Escherichia coli and Chlorobium tepidum. The structures show that the protein can be divided into three domains: a first FAD-binding domain showing the classical Rossmann fold, a second α/β domain inserted between two strands of the Rossmann fold, and an α-helical C-terminal domain. The domain inserted into the Rossmann fold displays structural similarity to the nicotinamide-adenine-dinucleotide-(phosphate)-binding domains of phenol hydroxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, and, correspondingly, we show that GidA binds NADH with high specificity as an initial donor of electrons. GidA behaves as a homodimer in solution. As revealed by the crystal structures, homodimerization is mediated via both the FAD-binding domain and the NADH-binding domain. Finally, a large patch of highly conserved, positively charged residues on the surface of GidA leading to the FAD-binding site suggests a tRNA-binding surface. We propose a model for the interaction between GidA and MnmE, which is supported by site-directed mutagenesis. Our data suggest that this interaction is modulated and potentially regulated by the switch function of the G domain of MnmE.     

The succinate dehydrogenase assembly factor,SdhE, is required for the flavinylation and activation of fumarate reductase in bacteria

The activity of the respiratory enzyme fumarate reductase (FRD) is dependent on the covalent attachment of the redox cofactor flavin adenine dinucleotide (FAD). We demonstrate that the FAD assembly factor SdhE, which flavinylates and activates the respiratory enzyme succinate dehydrogenase (SDH), is also required for the complete activation and flavinylation of FRD. SdhE interacted with, and flavinylated, the flavoprotein subunit FrdA, whilst mutations in a conserved RGxxE motif impaired the complete flavinylation and activation of FRD. These results are of widespread relevance because SDH and FRD play an important role in cellular energetics and are required for virulence in many important bacterial pathogens.     

The 2.3-A crystal structure of the shikimate 5-dehydrogenase orthologue YdiB from Escherichia coli suggests a novel catalytic environment for an NAD-dependent dehydrogenase

We present here the 2.3-A crystal structure of the Escherichia coli YdiB protein, an orthologue of shikimate 5-dehydrogenase. This enzyme catalyzes the reduction of 3-dehydroshikimate to shikimate as part of the shikimate pathway, which is absent in mammals but required for the de novo synthesis of aromatic amino acids, quinones, and folate in many other organisms. In this context, the shikimate pathway has been promoted as a target for the development of antimicrobial agents. The crystal structure of YdiB shows that the protomer contains two alpha/beta domains connected by two alpha-helices, with the N-terminal domain being novel and the C-terminal domain being a Rossmann fold. The NAD+ cofactor, which co-purified with the enzyme, is bound to the Rossmann domain in an elongated fashion with the nicotinamide ring in the pro-R conformation. Its binding site contains several unusual features, including a cysteine residue in close apposition to the nicotinamide ring and a clamp over the ribose of the adenosine moiety formed by phenylalanine and lysine residues. The structure explains the specificity for NAD versus NADP in different members of the shikimate dehydrogenase family on the basis of variations in the amino acid identity of several other residues in the vicinity of this ribose group. A cavity lined by residues that are 100% conserved among all shikimate dehydrogenases is found between the two domains of YdiB, in close proximity to the hydride acceptor site on the nicotinamide ring. Shikimate was modeled into this site in a geometry such that all of its heteroatoms form high quality hydrogen bonds with these invariant residues. Their strong conservation in all orthologues supports the possibility of developing broad spectrum inhibitors of this enzyme. The nature and disposition of the active site residues suggest a novel reaction mechanism in which an aspartate acts as the general acid/base catalyst during the hydride transfer reaction.     

Structure of the adenylation domain of an NAD+-dependent DNA ligase.

BACKGROUND: DNA ligases catalyse phosphodiester bond formation between adjacent bases in nicked DNA, thereby sealing the nick. A key step in the catalytic mechanism is the formation of an adenylated DNA intermediate. The adenyl group is derived from either ATP (in eucaryotes and archaea) or NAD+4 (in bacteria). This difference in cofactor specificity suggests that DNA ligase may be a useful antibiotic target. RESULTS: The crystal structure of the adenylation domain of the NAD+-dependent DNA ligase from Bacillus stearothermophilus has been determined at 2.8 A resolution. Despite a complete lack of detectable sequence similarity, the fold of the central core of this domain shares homology with the equivalent region of ATP-dependent DNA ligases, providing strong evidence for the location of the NAD+-binding site. CONCLUSIONS: Comparison of the structure of the NAD+4-dependent DNA ligase with that of ATP-dependent ligases and mRNA-capping enzymes demonstrates the manifold utilisation of a conserved nucleotidyltransferase domain within this family of enzymes. Whilst this conserved core domain retains a common mode of nucleotide binding and activation, it is the additional domains at the N terminus and/or the C terminus that provide the alternative specificities and functionalities in the different members of this enzyme superfamily.     

Crystal structures of ligand-bound saccharopine dehydrogenase from Saccharomyces cerevisiae

Three structures of saccharopine dehydrogenase (l-lysine-forming) (SDH) have been determined in the presence of sulfate, adenosine monophosphate (AMP), and oxalylglycine (OxGly). In the sulfate-bound structure, a sulfate ion binds in a cleft between the two domains of SDH, occupies one of the substrate carboxylate binding sites, and results in partial closure of the active site of the enzyme due to a domain rotation of almost 12 degrees in comparison to the apoenzyme structure. In the second structure, AMP binds to the active site in an area where the NAD+ cofactor is expected to bind. All of the AMP moieties (adenine ring, ribose, and phosphate) interact with specific residues of the enzyme. In the OxGly-bound structure, carboxylates of OxGly interact with arginine residues representative of the manner in which substrate (alpha-ketoglutarate and saccharopine) may bind. The alpha-keto group of OxGly interacts with Lys77 and His96, which are candidates for acid-base catalysis. Analysis of ligand-enzyme interactions, comparative structural analysis, corroboration with kinetic data, and discussion of a ternary complex model are presented in this study.     

New sequence motifs in flavoproteins: evidence for common ancestry and tools to predict structure

We describe two new sequence motifs, present in several families of flavoproteins. The "GG motif" (RxGGRxxS/T) is found shortly after the betaalphabetadinucleotide-binding motif (DBM) in L-amino acid oxidases, achacin and aplysianin-A, monoamine oxidases, corticosteroid-binding proteins, and tryptophan 2-monooxygenases. Other disperse sequence similarities between these families suggest a common origin. A GG motif is also found in protoporphyrinogen oxidase and carotenoid desaturases and, reduced to the central GG doublet, in the THI4 protein, dTDP-4-dehydrorhamnose reductase, soluble fumarate reductase, steroid dehydrogenases, Rab GDP-dissociation inhibitor, and in most flavoproteins with two dinucleotide-binding domains (glutathione reductase, glutamate synthase, flavin-containing monooxygenase, trimethylamine dehydrogenase...). In the latter families, an "ATG motif" (oxhhhATG) is found in both the FAD- and NAD(P)H-binding domains, forming the fourth beta-strand of the Rossman fold and the connecting loop. On the basis of these and previously described motifs, we present a classification of dinucleotide-binding proteins that could also serve as an evolutionary scheme. Like the DBM, the ATG motif appears to predate the divergence of NAD(P)H- and FAD-binding proteins. We propose that flavoproteins have evolved from a well-differentiated NAD(P)H-binding protein. The bulk of the substrate-binding domain was formed by an insertion after the fourth beta-strand, either of a closely related NAD(P)H-binding domain or of a domain of completely different origin.     

Modification of bovine heart succinate dehydrogenase with ethoxyformic anhydride and rose bengal: evidence for essential histidyl residues protectable by substrates

Purified and membrane-bound succinate dehydrogenase (SDH) from bovine heart mitochondria was inhibited by the histidine-modifying reagents ethoxyformic anhydride (EFA) and Rose Bengal in the presence of light. Succinate and competitive inhibitors protected against inhibition, and decreased the number of histidyl residues modified by EFA. The essential residue modified by EFA was not the essential thiol of SDH, but modification of the essential thiol abolished the protective effect of malonate against inhibition of SDH by EFA. The EFA inhibition was reversed by hydroxylamine nearly completely when the inhibition was less than or equal to 35%, and only partially when the inhibition was more extensive. The uv spectrum of EFA-modified SDH before and after hydroxylamine treatment suggested that extensive inhibition of SDH with EFA may result in ethoxyformylation at both imidazole nitrogens of histidyl residues. Such a modification is not reversed by hydroxylamine. Succinate dehydrogenases and fumarate reductases from several different sources have similar compositions, and the two enzymes from Escherichia coli have considerable homology in the amino acid composition of their respective flavoprotein and iron-sulfur protein subunits. In the former, there is a short stretch containing conserved histidine, cysteine, and arginine residues. These residues, if also conserved in the bovine enzyme, may be the essential active site residues suggested by this work (histidine) and previously (cysteine, arginine).     

Mechanism of coenzyme binding to human methionine synthase reductase revealed through the crystal structure of the FNR-like module and isothermal titration calorimetry

Human methionine synthase reductase (MSR) is a 78 kDa flavoprotein that regenerates the active form of cobalamin-dependent methionine synthase (MS). MSR contains one FAD and one FMN cofactor per polypeptide and functions in the sequential transfer of reducing equivalents from NADPH to MS via its flavin centers. We report the 1.9 A crystal structure of the NADP+-bound FNR-like module of MSR that spans the NADP(H)-binding domain, the FAD-binding domain, the connecting domain, and part of the extended hinge region, a feature unique to MSR. The overall fold of the protein is similar to that of the corresponding domains of the related diflavin reductase enzymes cytochrome P450 reductase and neuronal nitric oxide synthase (NOS). However, the extended hinge region of MSR, which is positioned between the NADP(H)/FAD- and FMN-binding domains, is in an unexpected orientation with potential implications for the mechanism of electron transfer. Compared with related flavoproteins, there is structural variation in the NADP(H)-binding site, in particular regarding those residues that interact with the 2'-phosphate and the pyrophosphate moiety of the coenzyme. The lack of a conserved binding determinant for the 2'-phosphate does not weaken the coenzyme specificity for NADP(H) over NAD(H), which is within the range expected for the diflavin oxidoreductase family of enzymes. Isothermal titration calorimetry reveals a binding constant of 37 and 2 microM for binding of NADP+ and 2',5'-ADP, respectively, for the ligand-protein complex formed with full-length MSR or the isolated FNR module. These values are consistent with Ki values (36 microM for NADP+ and 1.4 microM for 2',5'-ADP) obtained from steady-state inhibition studies. The relatively weaker binding of NADP+ to MSR compared with other members of the diflavin oxidoreductase family might arise from unique electrostatic repulsive forces near the 5'-pyrophosphate moiety and/or increased hydrophobic stacking between Trp697 and the re face of the FAD isoalloxazine ring. Small structural permutations within the NADP(H)-binding cleft have profound affects on coenzyme binding, which likely retards catalytic turnover of the enzyme in the cell. The biological implications of an attenuated mechanism of MS reactivation by MSR on methionine and folate metabolism are discussed.     

Probing domain mobility in a flavocytochrome

The crystal structures of various different members of the family of fumarate reductases and succinate dehydrogenases have allowed the identification of a mobile clamp (or capping) domain [e.g., Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], which has been proposed to be involved in regulating accessibility of the active site to substrate. To investigate this, we have constructed the A251C:S430C double mutant form of the soluble flavocytochrome c(3) fumarate reductase from Shewanella frigidimarina, to introduce an interdomain disulfide bond between the FAD-binding and clamp domains of the enzyme, thus restricting relative mobility between the two. Here, we describe the kinetic and crystallographic analysis of this double mutant enzyme. The 1.6 A resolution crystal structure of the A251C:S430C enzyme under oxidizing conditions reveals the formation of a disulfide bond, while Ellman analysis confirms its presence in the enzyme in solution. Kinetic analyses with the enzyme in both the nonbridged (free thiol) and the disulfide-bridged states indicate a slight decrease in the rate of fumarate reduction when the disulfide bridge is present, while solvent-kinetic-isotope studies indicate that in both wild-type and mutant enzymes the reaction is rate limited by proton and/or hydride transfer during catalysis. The limited effects of the inhibition of clamp domain mobility upon the catalytic reaction would indicate that such mobility is not essential for the regulation of substrate access or product release.     

Structure of Thermotoga maritima stationary phase survival protein SurE: a novel acid phosphatase.

BACKGROUND: The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase sigma subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. RESULTS: The structure of SurE from Thermotoga maritima was determined at 2.0 A. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability. CONCLUSIONS: The structure of SurE provided information about the protein's fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.     

Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 A resolution

Cholesterol oxidase (3 beta-hydroxysteroid oxidase, EC 1.1.3.6) is an FAD-dependent enzyme that carries out the oxidation and isomerization of steroids with a trans A : B ring junction. The crystal structure of the enzyme from Brevibacterium sterolicum has been determined using the method of isomorphous replacement and refined to 1.8 A resolution. The refined model includes 492 amino acid residues, the FAD prosthetic group and 453 solvent molecules. The crystallographic R-factor is 15.3% for all reflections between 10.0 A and 1.8 A resolution. The structure is made up of two domains: an FAD-binding domain and a steroid-binding domain. The FAD-binding domain consists of three non-continuous segments of sequence, including both the N terminus and the C terminus, and is made up of a six-stranded beta-sheet sandwiched between a four-stranded beta-sheet and three alpha-helices. The overall topology of this domain is very similar to other FAD-binding proteins. The steroid-binding domain consists of two non-continuous segments of sequence and contains a six-stranded antiparallel beta-sheet forming the "roof" of the active-site cavity. This large beta-sheet structure and the connections between the strands are topologically similar to the substrate-binding domain of the FAD-binding protein para-hydroxybenzoate hydroxylase. The active site lies at the interface of the two domains, in a large cavity filled with a well-ordered lattice of 13 solvent molecules. The flavin ring system of FAD lies on the "floor" of the cavity with N-5 of the ring system exposed. The ring system is twisted from a planar conformation by an angle of approximately 17 degrees, allowing hydrogen-bond interactions between the protein and the pyrimidine ring of FAD. The amino acid residues that line the active site are predominantly hydrophobic along the side of the cavity nearest the benzene ring of the flavin ring system, and are more hydrophilic on the opposite side near the pyrimidine ring. The cavity is buried inside the protein molecule, but three hydrophobic loops at the surface of the molecule show relatively high temperature factors, suggesting a flexible region that may form a possible path by which the substrate could enter the cavity. The active-site cavity contains one charged residue, Glu361, for which the side-chain electron density suggests a high degree of mobility for the side-chain. This residue is appropriately positioned to act as the proton acceptor in the proposed mechanism for the isomerization step.     

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA)

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.     

Crystal structure of the PIN domain of human telomerase-associated protein EST1A

Saccharomyces cerevisiae Est1p is a telomerase-associated protein essential for telomere length homeostasis. hEST1A is one of the three human Est1p homologues and is considered to be involved not only in regulation of telomere elongation or capping but also in nonsense-mediated degradation of RNA. hEST1A is composed of two conserved regions, Est1p homology and PIN (PilT N-terminus) domains. The present study shows the crystal structure of the PIN domain at 1.8 A resolution. The overall structure is composed of an alpha/beta fold or a core structure similar to the counterpart of 5' nucleases and an extended structure absent from archaeal PIN-domain proteins and 5' nucleases. The structural properties of the PIN domain indicate its putative active center consisting of invariant acidic amino acid residues, which is geometrically similar to the active center of 5' nucleases and an archaeal PAE2754 PIN-domain protein associated with exonuclease activity.     

Eukaryotic NAD+ synthetase Qns1 contains an essential,obligate intramolecular thiol glutamine amidotransferase domain related to nitrilase

NAD+ is an essential co-enzyme for redox reactions and is consumed in lysine deacetylation and poly(ADP-ribosyl)ation. NAD+ synthetase catalyzes the final step in NAD+ synthesis in the well characterized de novo, salvage, and import pathways. It has been long known that eukaryotic NAD+ synthetases use glutamine to amidate nicotinic acid adenine dinucleotide while many purified prokaryotic NAD+ synthetases are ammonia-dependent. Earlier, we discovered that glutamine-dependent NAD+ synthetases contain N-terminal domains that are members of the nitrilase superfamily and hypothesized that these domains function as glutamine amidotransferases for the associated synthetases. Here we show yeast glutamine-dependent NAD+ synthetase Qns1 requires both the nitrilase-related active-site residues and the NAD+ synthetase active-site residues for function in vivo. Despite failure to complement the lethal phenotype of qns1 disruption, the former mutants retain ammonia-dependent NAD+ synthetase activity in vitro, whereas the latter mutants retain basal glutaminase activity. Moreover, the two classes of mutants fail to trans-complement despite forming a stable heteromultimer in vivo. These data indicate that the nitrilase-related domain in Qns1 is the fourth independently evolved glutamine amidotransferase domain to have been identified in nature and that glutamine-dependence is an obligate phenomenon involving intramolecular transfer of ammonia over a predicted distance of 46 A from one active site to another within Qns1 monomers.     

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.