Comparison of the Intrinsic Dynamics of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) are an important family of enzymes that catalyze tRNA aminoacylation reaction (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. AARSs are grouped into two broad classes (class I and II) based on sequence/structural homology and mode of their interactions with the tRNA molecule (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. As protein dynamics play an important role in enzyme function, we explored the intrinsic dynamics of these enzymes using normal mode analysis and investigated if the two classes and six subclasses (Ia–c and IIa–c) of AARSs exhibit any distinct patterns of motion. The present study found that the intrinsic dynamics-based classification of these enzymes is similar to that obtained based on sequence/structural homology for most enzymes. However, the classification of seryl-tRNA synthetase was not straightforward; the internal mobility patterns of this enzyme are comparable to both IIa and IIb AARSs. This study revealed only a few general mobility patterns in these enzymes—(1) the insertion domain is generally engaged in anticorrelated motion with respect to the catalytic domain for both classes of AARSs and (2) anticodon binding domain dynamics are partly correlated and partly anticorrelated with respect to other domains for class I enzymes. In most of the class II AARSs, the anticodon binding domain is predominately engaged in anticorrelated motion with respect to the catalytic domain and correlated to the insertion domain. This study supports the notion that dynamic-based classification could be useful for functional classification of proteins.     

A conserved nuclease domain in the archaeal Holliday junction resolving enzyme Hjc

Holliday junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing the rearrangement and repair of DNA. They are metal ion-dependent endonucleases with strong structural specificity for branched DNA species. Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday junction resolving enzyme, Hjc, has recently been identified. We demonstrate that Hjc manipulates the global structure of the Holliday junction into a 2-fold symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA 3' to the point of strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that has been identified previously in several restriction enzymes. The roles of catalytic residues conserved within this domain have been confirmed by site-directed mutagenesis. This is the first example of this domain in an archaeal enzyme of known function as well as the first in a Holliday junction resolving enzyme.     

Crystal structure of prephenate dehydrogenase from Aquifex aeolicus. Insights into the catalytic mechanism

The enzyme prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. Prephenate dehydrogenases exist as either monofunctional or bifunctional enzymes. The bifunctional enzymes are diverse, since the prephenate dehydrogenase domain is associated with other enzymes, such as chorismate mutase and 3-phosphoskimate 1-carboxyvinyltransferase. We report the first crystal structure of a monofunctional prephenate dehydrogenase enzyme from the hyper-thermophile Aquifex aeolicus in complex with NAD+. This protein consists of two structural domains, a modified nucleotide-binding domain and a novel helical prephenate binding domain. The active site of prephenate dehydrogenase is formed at the domain interface and is shared between the subunits of the dimer. We infer from the structure that access to the active site is regulated via a gated mechanism, which is modulated by an ionic network involving a conserved arginine, Arg250. In addition, the crystal structure reveals for the first time the positions of a number of key catalytic residues and the identity of other active site residues that may participate in the reaction mechanism; these residues include Ser126 and Lys246 and the catalytic histidine, His147. Analysis of the structure further reveals that two secondary structure elements, beta3 and beta7, are missing in the prephenate dehydrogenase domain of the bifunctional chorismate mutase-prephenate dehydrogenase enzymes. This observation suggests that the two functional domains of chorismate mutase-prephenate dehydrogenase are interdependent and explains why these domains cannot be separated.     

Nucleotide pyrophosphatases/phosphodiesterases on the move

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) release nucleoside 5'-monophosphates from nucleotides and their derivatives. They exist both as membrane proteins, with an extracellular active site, and as soluble proteins in body fluids. The only well-characterized NPPs are the mammalian ecto-enzymes NPP1 (PC-1), NPP2 (autotaxin) and NPP3 (B10; gp130(RB13-6)). These are modular proteins consisting of a short N-terminal intracellular domain, a single transmembrane domain, two somatomedin-B-like domains, a catalytic domain, and a C-terminal nuclease-like domain. The catalytic domain of NPPs is conserved from prokaryotes to mammals and shows remarkable structural and catalytic similarities with the catalytic domain of other phospho-/sulfo-coordinating enzymes such as alkaline phosphatases. Hydrolysis of pyrophosphate/phosphodiester bonds by NPPs occurs via a nucleotidylated threonine. NPPs are also known to auto(de)phosphorylate this active-site threonine, a process accounted for by an intrinsic phosphatase activity, with the phosphorylated enzyme representing the catalytic intermediate of the phosphatase reaction. NPP1-3 have been implicated in various processes, including bone mineralization, signaling by insulin and by nucleotides, and the differentiation and motility of cells. While it has been established that most of these biological effects of NPPs require a functional catalytic site, their physiological substrates remain to be identified.     

Tight association of N-terminal and catalytic subunits of rabbit 12/15-lipoxygenase is important for protein stability and catalytic activity

12/15-Lipoxygenases (12/15-LOXs) have been implicated in inflammatory and hyperproliferative diseases but the structural biology of these enzymes is not well developed. Most LOXs constitute single polypeptide chain proteins that fold into a two-domain structure. In the crystal structure the two domains are tightly associated, but small angle X-ray scattering data and dynamic fluorescence studies suggested a high degree of structural flexibility involving movement of the N-terminal domain relative to catalytic subunit. When we inspected the interdomain interface we have found a limited number of side-chain contacts which are involved in interactions of these two structural subunits. One of such contact points involves tyrosine 98 of N-terminal domain. This aromatic amino acid is invariant in vertebrate LOXs regardless of overall sequence identity. To explore in more detail the role of aromatic interactions in interdomain association we have mutated Y98 to various residues and quantified the structural and functional consequences of these alterations. We have found that loss of an aromatic moiety at position 98 impaired the catalytic activity and membrane binding capacity of the mutant enzymes. Although CD and fluorescence emission spectra of wild-type and mutant enzyme species were indistinguishable, the mutation led to enlargement of the molecular shape of the enzyme as detected by analytic gel filtration and this structural alteration was shown to be associated with a loss of protein thermal stability. The possible role of tight interdomain association for the enzyme's structural performance is discussed.     

Mutational analysis of the Mycobacterium tuberculosis Rv1625c adenylyl cyclase: residues that confer nucleotide specificity contribute to dimerization

The mycobacterial Rv1625c gene product is an adenylyl cyclase with sequence similarity to the mammalian enzymes. The catalytic domain of the enzyme forms a homodimer and residues specifying adenosine triphosphate (ATP) specificity lie at the dimer interface. Mutation of these residues to those present in guanylyl cyclases failed to convert the enzyme to a guanylyl cyclase, but dramatically reduced its adenylyl cyclase activity and altered its oligomeric state. Computational modeling revealed subtle differences in the dimer interface that could explain the biochemical data, suggesting that the structural and catalytic features of this homodimeric adenylyl cyclase are in contrast to those of the heterodimeric mammalian enzymes.     

Nucleotide Pyrophosphatases/Phosphodiesterases on the Move

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) release nucleoside 5′-monophosphates from nucleotides and their derivatives. They exist both as membrane proteins, with an extracellular active site, and as soluble proteins in body fluids. The only well-characterized NPPs are the mammalian ecto-enzymes NPP1 (PC-1), NPP2 (autotaxin) and NPP3 (B10; gp130^RB13-6). These are modular proteins consisting of a short N-terminal intracellular domain, a single transmembrane domain, two somatomedin-B-like domains, a catalytic domain, and a C-terminal nuclease-like domain. The catalytic domain of NPPs is conserved from prokaryotes to mammals and shows remarkable structural and catalytic similarities with the catalytic domain of other phospho-/sulfo-coordinating enzymes such as alkaline phosphatases. Hydrolysis of pyrophosphate/phosphodiester bonds by NPPs occurs via a nucleotidylated threonine. NPPs are also known to auto(de)phosphorylate this active-site threonine, a process accounted for by an intrinsic phosphatase activity, with the phosphorylated enzyme representing the catalytic intermediate of the phosphatase reaction. NPP1-3 have been implicated in various processes, including bone mineralization, signaling by insulin and by nucleotides, and the differentiation and motility of cells. While it has been established that most of these biological effects of NPPs require a functional catalytic site, their physiological substrates remain to be identified.     

Sequence and structural differences between enzyme and nonenzyme homologs

To improve our understanding of the evolution of novel functions, we performed a sequence, structural, and functional analysis of homologous enzymes and nonenzymes of known three-dimensional structure. In most examples identified, the nonenzyme is derived from an ancestral catalytic precursor (as opposed to the reverse evolutionary scenario, nonenzyme to enzyme), and the active site pocket has been disrupted in some way, owing to the substitution of critical catalytic residues and/or steric interactions that impede substrate binding and catalysis. Pairwise sequence identity is typically insignificant, and almost one-half of the enzyme and nonenzyme pairs do not share any similarity in function. Heterooligomeric enzymes comprising homologous subunits in which one chain is catalytically inactive and enzyme polypeptides that contain internal catalytic and noncatalytic duplications of an ancient enzyme domain are also discussed.     

Glucoamylase from Thermoanaerobacterium thermosaccharolyticum: Sequence studies and analysis of the macromolecular architecture of the enzyme

A chromosomal DNA fragment with a length of 2,025 bp, carrying the structural gene coding for glucoamylase in Thermoanaerobacterium thermosaccharolyticum, was cloned and sequenced. It coded for 695 amino acids, representing a polypeptide with a predicted molecular mass of 77.5 kDa. The deduced amino acid sequence exhibited high homologies with the glucoamylase sequence of another bacterial glucoamylase (Clostridium sp. G0005) and with fungal glucoamylases. The catalytic domain (amino acids 271 to 695) of the T. thermosaccharolyticum enzyme shared a high degree of similarity (five conserved regions) with the catalytic domain of Aspergillus awamori glucoamylase. By comparing the secondary structure of the sequence of the catalytic domain of the T. thermosaccharolyticum enzyme with that of glucoamylase from A. awamori, and on the basis of X-ray crystallographic data available for the A. awamori enzyme, it turned out that, most probably, both enzymes have a catalytic domain organized into an "(alpha/alpha)(6)-barrel" and an overall size and shape that is very similar. These findings confirm and extend our working model for the macromolecular architecture of the T. thermosaccharolyticum glucoamylase obtained, in earlier experiments, by electron microscopy of negatively stained isolated enzyme molecules. Antibodies for an enzyme-specific peptide located near the active site were successfully applied for inhibition studies of enzyme activity and for electron microscopic epitope mapping. A study comparing the site of attachment of this kind of antibody to the T. thermosaccharolyticum glucoamylase molecule with the expected attachment site as deduced from the A. awamori enzyme structure confirmed the close similarity of both glucoamylases regarding the macromolecular architecture of that part of the enzyme carrying the catalytic center, though helices H9, H10, and H11 in peripheral parts of the A. awamori enzyme are missing in the T. thermosaccharolyticum enzyme.     

The structure of a GH149 β-(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide-specific GH94 glucose-β-(1 → 3)-glucose (laminaribiose) phosphorylase

Glycoside phosphorylases (GPs) with specificity for β-(1 → 3)-gluco-oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far—glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X-ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β-(1 → 3)-gluco-oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes.     

Contrasting catalytic and allosteric mechanisms for phosphoglycerate dehydrogenases

D-3-Phosphoglycerate dehydrogenases (PGDH) exist with at least three different structural motifs and the enzymes from different species display distinctly different mechanisms. In many species, particularly bacteria, the catalytic activity is regulated allosterically through binding of l-serine to a distinct structural domain, termed the ACT domain. Some species, such as Mycobacterium tuberculosis, contain an additional domain, called the "allosteric substrate binding" or ASB domain, that functions as a co-domain in the regulation of catalytic activity. That is, both substrate and effector function synergistically in the regulation of activity to give the enzyme some interesting properties that may have physiological relevance for the persistent state of tuberculosis. Both enzymes function through a V-type regulatory mechanism and, in the Escherichia coli enzyme, it has been demonstrated that this results from a dead-end complex that decreases the concentration of active species rather than a decrease in the velocity of the active species. This review compares and contrasts what we know about these enzymes and provides additional insight into their mechanism of allosteric regulation.     

Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases

Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements.     

Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP: MECHANISTIC INSIGHTS INTO A MINIMALISTIC E1 ENZYME*

E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys²⁵⁰) is part of the adenylation domain in an α-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.     

Structural features of the Nostoc punctiforme debranching enzyme reveal the basis of its mechanism and substrate specificity

The debranching enzyme Nostoc punctiforme debranching enzyme (NPDE) from the cyanobacterium Nostoc punctiforme (PCC73102) hydrolyzes the α‐1,6 glycosidic linkages of malto‐oligosaccharides. Despite its high homology to cyclodextrin/pullulan (CD/PUL)‐hydrolyzing enzymes from glycosyl hydrolase 13 family (GH‐13), NPDE exhibits a unique catalytic preference for longer malto‐oligosaccharides (>G8), performing hydrolysis without the transgylcosylation or CD‐hydrolyzing activities of other GH‐13 enzymes. To investigate the molecular basis for the property of NPDE, we determined the structure of NPDE at 2.37‐Å resolution. NPDE lacks the typical N‐terminal domain of other CD/PUL‐hydrolyzing enzymes and forms an elongated dimer in a head‐to‐head configuration. The unique orientation of residues 25–55 in NPDE yields an extended substrate binding groove from the catalytic center to the dimeric interface. The substrate binding groove with a lengthy cavity beyond the ?1 subsite exhibits a suitable architecture for binding longer malto‐oligosaccharides (>G8). These structural results may provide a molecular basis for the substrate specificity and catalytic function of this cyanobacterial enzyme, distinguishing it from the classical neopullulanases and CD/PUL‐hydrolyzing enzymes. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Crystal structure of a fragment of mouse ubiquitin-activating enzyme

Protein ubiquitination requires the sequential activity of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligase (E3). The ubiquitin-transfer machinery is hierarchically organized; for every ubiquitin-activating enzyme, there are several ubiquitin-conjugating enzymes, and most ubiquitin-conjugating enzymes can in turn interact with multiple ubiquitin ligases. Despite the central role of ubiquitin-activating enzyme in this cascade, a crystal structure of a ubiquitin-activating enzyme is not available. The enzyme is thought to consist of an adenylation domain, a catalytic cysteine domain, a four-helix bundle, and possibly, a ubiquitin-like domain. Its adenylation domain can be modeled because it is clearly homologous to the structurally known adenylation domains of the activating enzymes for the small ubiquitin-like modifier (SUMO) and for the protein encoded by the neuronal precursor cell-expressed, developmentally down-regulated gene 8 (NEDD8). Low sequence similarity and vastly different domain lengths make modeling difficult for the catalytic cysteine domain that results from the juxtaposition of two catalytic cysteine half-domains. Here, we present a biochemical and crystallographic characterization of the two half-domains and the crystal structure of the larger, second catalytic cysteine half-domain of mouse ubiquitin-activating enzyme. We show that the domain is organized around a conserved folding motif that is also present in the NEDD8- and SUMO-activating enzymes, and we propose a tentative model for full-length ubiquitin-activating enzyme.     

Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.     

Crystal structure of human mitochondrial acyl-CoA thioesterase (ACOT2)

Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of CoA esters to free CoA and carboxylic acids and have important functions in lipid metabolism and other cellular processes. Type I ACOTs are found only in animals and contain an α/β hydrolase domain, through currently no structural information is available on any of these enzymes. We report here the crystal structure at 2.1 Å resolution of human mitochondrial ACOT2, a type I enzyme. The structure contains two domains, N and C domains. The C domain has the α/β hydrolase fold, with the catalytic triad Ser294-His422-Asp388. The N domain contains a seven-stranded β-sandwich, which has some distant structural homologs in other proteins. The active site is located in a large pocket at the interface between the two domains. The structural information has significant relevance for other type I ACOTs and related enzymes.     

Identification of essential residues for catalysis of rat intestinal phospholipase B/lipase

Intestinal brush border membrane-associated phospholipase B/lipase (PLB/LIP) consists of four tandem homologous domains (repeats 1 through 4) and a COOH-terminal membrane binding domain, and repeat 2 is the catalytic domain that catalyzes phospholipase A2, lysophospholipase, and lipase activities. We examined the structural basis of the catalysis of PLB/LIP with this unique substrate specificity by site-directed mutagenesis of recombinant repeat 2 enzyme. Ser414 and Ser459 within the active serine-containing consensus sequence G-X-S-X-G in the best-established lipase family were dispensable for activity. In contrast, substitution of Ala for Ser404 almost completely inactivated the three lipolytic activities of PLB/LIP, even though the gross conformation was not altered as determined by CD spectroscopy. Notably, this Ser is located within the conserved G-D-S-L sequence on the NH2-terminal side in lipolytic enzymes of another group proposed recently. Furthermore, mutagenesis and CD spectroscopic analyses suggested that Asp518 and His659, lying within conserved short stretches in the latter group of lipolytic enzymes, were essential for activity. These three essential residues are conserved in the known PLB/LIP enzymes, suggesting that they form the catalytic triad in the active site. These results indicate that PLB/LIP represents a distinct class of the lipase family. PLB/LIP is the first mammalian member of that family. Repeat 2 is equipped with the triad, but not the other repeats, accounting for why only repeat 2 is the catalytic domain. Replacing Thr406 with Gly, matching the enzyme's sequence to the lipase consensus sequence exactly, led to a great decrease in secretion and accumulation of inactive enzyme in the cells, suggesting a role of Thr406 in the structural stability.