Crystal structure of a theta-class glutathione transferase.

Glutathione S-transferases (GSTs) are a family of enzymes involved in the cellular detoxification of xenotoxins. Cytosolic GSTs have been grouped into four evolutionary classes for which there are representative crystal structures of three of them. Here we report the first crystal structure of a theta-class GST. So far, all available GST crystal structures suggest that a strictly conserved tyrosine near the N-terminus plays a critical role in the reaction mechanism and such a role has been convincingly demonstrated by site-directed mutagenesis. Surprisingly, the equivalent residue in the theta-class structure is not in the active site, but its role appears to have been replaced by either a nearby serine or by another tyrosine residue located in the C-terminal domain of the enzyme.     

Cloning,expression, and characterization of a human 4'-phosphopantetheinyl transferase with broad substrate specificity

A single candidate 4'-phosphopantetheine transferase, identified by BLAST searches of the human genome sequence data base, has been cloned, expressed, and characterized. The human enzyme, which is expressed mainly in the cytosolic compartment in a wide range of tissues, is a 329-residue, monomeric protein. The enzyme is capable of transferring the 4'-phosphopantetheine moiety of coenzyme A to a conserved serine residue in both the acyl carrier protein domain of the human cytosolic multifunctional fatty acid synthase and the acyl carrier protein associated independently with human mitochondria. The human 4'-phosphopantetheine transferase is also capable of phosphopantetheinylation of peptidyl carrier and acyl carrier proteins from prokaryotes. The same human protein also has recently been implicated in phosphopantetheinylation of the alpha-aminoadipate semialdehyde dehydrogenase involved in lysine catabolism (Praphanphoj, V., Sacksteder, K. A., Gould, S. J., Thomas, G. H., and Geraghty, M. T. (2001) Mol. Genet. Metab. 72, 336-342). Thus, in contrast to yeast, which utilizes separate 4'-phosphopantetheine transferases to service each of three different carrier protein substrates, humans appear to utilize a single, broad specificity enzyme for all posttranslational 4'-phosphopantetheinylation reactions.     

Crystal structure of YHI9, the yeast member of the phenazine biosynthesis PhzF enzyme superfamily

In the Pseudomonas bacterial genomes, the PhzF proteins are involved in the production of phenazine derivative antibiotic and antifungal compounds. The PhzF superfamily however also encompasses proteins in all genomes from bacteria to eukaryotes, for which no function has been assigned. We have determined the three dimensional crystal structure at 2.05 A resolution of YHI9, the yeast member of the PhzF family. YHI9 has a fold similar to bacterial diaminopimelate epimerase, revealing a bimodular structure with an internal symmetry. Residue conservation identifies a putative active site at the interface between the two domains. Evolution of this protein by gene duplication, gene fusion and domain swapping from an ancestral gene containing the "hot dog" fold, identifies the protein as a "kinked double hot dog" fold.     

Crystal structure of the nucleotide‐metabolizing enzyme NTPDase4

The ecto‐nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes found on the cell surface and in the lumen of certain organelles, that are major regulators of purinergic signaling. Their intracellular roles, however, have not been clearly defined. NTPDase4 (UDPase, ENTPD4) is a Golgi protein potentially involved in nucleotide recycling as part of protein glycosylation, and is also found in lysosomes, where its purpose is unknown. To further our understanding of NTPDase4 function, we determined its crystal structure. The enzyme adopts a wide open, inactive conformation. Differences in the nucleotide‐binding site relative to its homologs could account for its substrate selectivity. The putative membrane‐interacting loop of cell‐surface NTPDases is drastically altered in NTPDase4, potentially affecting its interdomain dynamics at the Golgi membrane.     

Crystal structure of human thioesterase superfamily member 2

Hotdog-fold has been identified in more than 1000 proteins, yet many of which in eukaryotes are less studied. No structural or functional studies of human thioesterase superfamily member 2 (hTHEM2) have been reported before. Since hTHEM2 exhibits about 20% sequence identity to Escherichia coli PaaI protein, it was proposed to be a thioesterase with a hotdog-fold. Here, we report the crystallographic structure of recombinant hTHEM2, determined by the single-wavelength anomalous dispersion method at 2.3A resolution. This structure demonstrates that hTHEM2 indeed contains a hotdog-fold and forms a back-to-back tetramer as other hotdog proteins. Based on structural and sequence conservation, the thioesterase active site in hTHEM2 is predicted. The structure and substrate specificity are most similar to those of the bacterial phenylacetyl-CoA hydrolase. Asp65, located on the central alpha-helix of subunit B, was shown by site-directed mutagenesis to be essential to catalysis.     

Crystal structure of UDP-galactose 4-epimerase-like L-threonine dehydrogenase belonging to the intermediate short-chain dehydrogenase-reductase superfamily

The crystal structure of a L-threonine dehydrogenase (L-ThrDH; EC 1.1.1.103) from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1, which shows no sequence similarity to conventional L-ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol. The asymmetric unit consisted of two subunits related by a two-fold rotation axis. Each monomer consisted of a Rossmann-fold domain and a carboxyl-terminal catalytic domain. The overall fold of F. frigidimaris L-ThrDH showed significant similarity to that of UDP-galactose 4-epimerase (GalE); however, structural comparison of the enzyme with E. coli and human GalEs showed clear topological differences in three loops (loop 1, loop 2 and the NAD-binding loop) around the substrate and NAD binding sites. In F. frigidimaris L-ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier preventing the binding of UDP-glucose. Alternatively, loop 1 contributes to a unique substrate binding pocket in the F. frigidimaris enzyme. The NAD binding loop, which tightly holds the adenine ribose moiety of NAD in the Escherichia coli and human GalEs, is absent in F. frigidimaris L-ThrDH. Consequently, the cofactor binds to F. frigidimaris L-ThrDH in a reversible manner, unlike its binding to GalE. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of L-threonine via either a proton shuttle mechanism driven by Tyr143 and facilitated by Ser118 or direct proton transfer driven by Tyr143. The present structure provides a clear bench mark for distinguishing GalE-like L-ThrDHs from GalEs.     

Crystal structure of CHO reductase, a member of the aldo-keto reductase superfamily

Chinese hamster ovary (CHO) reductase is an enzyme belonging to the aldo-keto reductase (AKR) superfamily that is induced by the aldehyde-containing protease inhibitor ALLN (Inoue, Sharma, Schimke, et al., J Biol Chem 1993;268: 5894). It shows 70% sequence identity to human aldose reductase (Hyndman, Takenoshita, Vera, et al., J Biol Chem 1997;272:13286), which is a target for drug design because of its implication in diabetic complications. We have determined the crystal structure of CHO reductase complexed with nicotinamide adenine dinucleotide phosphate (NADP)+ to 2.4 A resolution. Similar to aldose reductase and other AKRs, CHO reductase is an alpha/beta TIM barrel enzyme with cofactor bound in an extended conformation. All key residues involved in cofactor binding are conserved with respect to other AKR members. CHO reductase shows a high degree of sequence identity (91%) with another AKR member, FR-1 (mouse fibroblast growth factor-regulated protein), especially around the variable C-terminal end of the protein and has a similar substrate binding pocket that is larger than that of aldose reductase. However, there are distinct differences that can account for differences in substrate specificity. Trp111, which lies horizontal to the substrate pocket in all other AKR members is perpendicular in CHO reductase and is accompanied by movement of Leu300. This coupled with movement of loops A, B, and C away from the active site region accounts for the ability of CHO reductase to bind larger substrates. The position of Trp219 is significantly altered with respect to aldose reductase and appears to release Cys298 from steric constraints. These studies show that AKRs such as CHO reductase are excellent models for examining the effects of subtle changes in amino acid sequence and alignment on binding and catalysis.     

Crystal structure of brain pyridoxal kinase,a novel member of the ribokinase superfamily

The three-dimensional structures of brain pyridoxal kinase and its complex with the nucleotide ATP have been elucidated in the dimeric form at 2.1 and 2.6 A, respectively. Results have shown that pyridoxal kinase, as an enzyme obeying random sequential kinetics in catalysis, does not possess a lid shape structure common to all kinases in the ribokinase superfamily. This finding has been shown to be in line with the condition that pyridoxal kinase binds substrates with variable sizes of chemical groups at position 4 of vitamin B(6) and its derivatives. In addition, the enzyme contains a 12-residue peptide loop in the active site for the prevention of premature hydrolysis of ATP. Conserved amino acid residues Asp(118) and Tyr(127) in the peptide loop could be moved to a position covering the nucleotide after its binding so that its chance to hydrolyze in the aqueous environment of the active site was reduced. With respect to the evolutionary trend of kinase enzymes, the existence of this loop in pyridoxal kinase could be classified as an independent category in the ribokinase superfamily according to the structural feature found and mechanism followed in catalysis.     

Crystal structure of Bacillus subtilis guanine deaminase: the first domain-swapped structure in the cytidine deaminase superfamily

Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-A resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 A(2) per monomer. The essential zinc ion is ligated by a water molecule together with His(53), Cys(83), and Cys(86). A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp(114) and Tyr(156'). The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases.     

Crystal structure of the superfamily 1 helicase from Tomato mosaic virus

The genomes of the Tomato mosaic virus and many other plant and animal positive-strand RNA viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato mosaic virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/β domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded β-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for tomato mosaic virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.     

Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis

The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzyme dihydroflavonol 4-reductase (DFR) catalyzes a late step in the biosynthesis of anthocyanins and condensed tannins, two flavonoid classes of importance to plant survival and human nutrition. This enzyme has been widely investigated in many plant species, but little is known about its structural and biochemical properties. To provide a basis for detailed structure-function studies, the crystal structure of Vitis vinifera DFR, heterologously expressed in Escherichia coli, has been determined at 1.8 Å resolution. The 3D structure of the ternary complex obtained with the oxidized form of nicotinamide adenine dinucleotide phosphate and dihydroquercetin, one of the DFR substrates, presents common features with the short-chain dehydrogenase/reductase family, i.e., an N-terminal domain adopting a Rossmann fold and a variable C-terminal domain, which participates in substrate binding. The structure confirms the importance of the 131-156 region, which lines the substrate binding site and enlightens the role of a specific residue at position 133 (Asn or Asp), assumed to control substrate recognition. The activity of the wild-type enzyme and its variant N133D has been quantified in vitro, using dihydroquercetin or dihydrokaempferol. Our results demonstrate that position 133 cannot be solely responsible for the recognition of the B-ring hydroxylation pattern of dihydroflavonols.     

Crystal structure of CaiB, a type-III CoA transferase in carnitine metabolism

Carnitine is an important molecule in human metabolism, mainly because of its role in the transport of long-chain fatty acids across the inner mitochondrial membrane. Escherichia coli uses carnitine as a terminal electron acceptor during anaerobic metabolism. Bacteria present in our large intestine break down carnitine that is not absorbed in the small intestine. One part of this catabolic pathway is reversible and can be utilized for bioproduction of large amounts of stereochemically pure L-carnitine, which is used medically for the treatment of a variety of human diseases. Here, we present the crystal structure of the E. coli protein CaiB, which is a member of the recently identified type-III coenzyme A (CoA) transferase family and catalyzes the transfer of the CoA moiety between gamma-butyrobetaine-CoA and carnitine forming carnityl-CoA and gamma-butyrobetaine. This is the first protein from the carnitine metabolic pathway to be structurally characterized. The structure of CaiB reveals a spectacular fold where two monomers are interlaced to form an interlocked dimer. A molecule of the crystallization buffer bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (bis-tris) is bound in a large pocket located primarily in the small domain, and we propose that this pocket constitutes the binding site for both substrate moieties participating in the CaiB transfer reaction. The binding of CoA to CaiB induces a domain movement that closes the active site of the protein. This is the first observation of a domain movement in the type-III CoA transferase family and can play an important role in coupling substrate binding to initiation of the catalytic reaction.     

Functional characterization of 4'-phosphopantetheinyl transferase genes of bacterial and fungal origin by complementation of Saccharomyces cerevisiae lys5

Lysine biosynthesis in yeast requires the posttranslational conversion of the alpha-aminoadipate semialdehyde reductase Lys2 by the 4'-phosphopantetheinyl transferase (PPTase) Lys5 from the inactive apo-form into the catalytically active holo-form. In this reaction, the peptidyl carrier domain of Lys2 is modified at a conserved serine residue side chain with the 4'-phosphopantetheine moiety derived from coenzyme A. We have deleted the lys5 gene in Saccharomyces cerevisiae to investigate the substrate specificity of various heterologous PPTase genes of bacterial and fungal origin by testing their ability to complement lys5 in trans. Genes encoding PPTases Sfp and Gsp from Bacillus spp., which are involved in non-ribosomal peptide antibiotic synthesis, complemented the lys5 deletion, whereas ydcB of Bacillus subtilis, which encodes the acyl carrier protein synthase involved in fatty acid synthesis, could not. Two yet uncharacterized fungal genes, q10474 of Schizosaccharomyces pombe, meanwhile annotated as the putative lys7 gene, and npgA of Aspergillus nidulans, also complemented the lys5 deletion and have thus been functionally characterized as PPTases. The complementation system described also provides the basis for a simple method of functional characterization of PPTase candidate genes and their cloning from chromosomal DNA or cDNA libraries of diverse origin.     

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.     

A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product biosynthesis genes from metagenome libraries

The single-module non-ribosomal peptide synthetase BpsA from Streptomyces lavendulae has the unique ability to autonomously synthesize a coloured product (indigoidine) from a single substrate (l-glutamine), conditional upon activation by a 4'-phosphopantetheinyl transferase (PPTase) partner. We show that bpsA can be expressed in an entD PPTase gene deleted mutant of Escherichia coli to yield a sensitive reporter strain for recovery of PPTase genes from metagenome libraries. We also show that recombinant bpsA constructs, generated by substitution of the native peptidyl carrier protein domain followed by directed evolution to restore function, can be used to increase the diversity of PPTase genes recovered from a sample. As PPTases are essential for activation of non-ribosomal peptide synthetase and polyketide synthase enzymes, they are frequently associated with secondary metabolite gene clusters. Nearly half of the PPTases recovered in our screening of two small-insert soil metagenome libraries were genetically linked to recognizable secondary metabolite biosynthetic genes, demonstrating that PPTase-targeting functional screens can be used for efficient recovery of natural product gene clusters from metagenome libraries. The plasticity and portability of bpsA reporter genes can potentially be exploited to maximize recovery and expression of PPTase-bearing clones in a wide range of hosts.