High resolution x-ray structure of tyvelose epimerase from Salmonella typhi

Tyvelose epimerase catalyzes the last step in the biosynthesis of tyvelose by converting CDP-d-paratose to CDP-d-tyvelose. This unusual 3,6-dideoxyhexose occurs in the O-antigens of some types of Gram-negative bacteria. Here we describe the cloning, protein purification, and high-resolution x-ray crystallographic analysis of tyvelose epimerase from Salmonella typhi complexed with CDP. The enzyme from S. typhi is a homotetramer with each subunit containing 339 amino acid residues and a tightly bound NAD+ cofactor. The quaternary structure of the enzyme displays 222 symmetry and can be aptly described as a dimer of dimers. Each subunit folds into two distinct lobes: the N-terminal motif responsible for NAD+ binding and the C-terminal region that harbors the binding site for CDP. The analysis described here demonstrates that tyvelose epimerase belongs to the short-chain dehydrogenase/reductase superfamily of enzymes. Indeed, its active site is reminiscent to that observed for UDP-galactose 4-epimerase, an enzyme that plays a key role in galactose metabolism. Unlike UDP-galactose 4-epimerase where the conversion of configuration occurs about C-4 of the UDP-glucose or UDP-galactose substrates, in the reaction catalyzed by tyvelose epimerase, the inversion of stereochemistry occurs at C-2. On the basis of the observed binding mode for CDP, it is possible to predict the manner in which the substrate, CDP-paratose, and the product, CDP-tyvelose, might be accommodated within the active site of tyvelose epimerase.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: analysis of its hysteretic behavior during catalysis

UDP-galactose 4-epimerase serves as a prototype model of class II oxidoreductases that use bound NAD as a cofactor. This enzyme from Kluyveromyces fragilis is a homodimer with a molecular mass of 75 kDa/subunit. Continuous monitoring of the conversion of UDP-galactose (UDP-gal) to UDP-glucose (UDP-glu) by the epimerase in the presence of the coupling enzyme UDP-glucose dehydrogenase and NAD shows a kinetic lag of up to 80 s before a steady state is reached. The disappearance of the lag follows first-order kinetics (k = 3.22 x 10(-2) s(-1)) at 25 degrees C at enzyme and substrate concentrations of 1.0 nM and 1 mM, respectively. The observed lag is not due to factors such as insufficient activity of the coupling enzyme, association or dissociation or incomplete recruitment of NAD by epimerase, product activation, etc., but was a true expression of the activity of the prepared enzyme. Dissociation of the bound ligand(s) by heat followed by analysis with reverse-phase HPLC, TLC, UV-absorption spectrometry, mass spectrometry, and NMR showed that in addition to 1.78 mol of NAD/dimer, the epimerase also contains 0.77 mol of 5'-UMP/dimer. The latter is a strong competitive inhibitor. Preincubation of the epimerase with the substrate UDP-gal or UDP-glu replaces the inhibitor and also abolishes the lag, which reappeared after the enzyme was treated with 5'-UMP. The lag was not observed as long as the cells were in the growing phase and galactose in the growth medium was limiting, suggesting that association with 5'-UMP is a late log-phase phenomenon. The stoichiometry and conserved amino acid sequence around the NAD binding site of multimeric class I (classical dehydrogenases) and class II oxidoreductases, as reported in the literature, have been compared. It shows that each subunit is independently capable of being associated with one molecule of NAD, suggestive of two NAD binding sites of epimerase per dimer.     

13C NMR analysis of electrostatic interactions between NAD+ and active site residues of UDP-galactose 4-epimerase: implications for the activation induced by uridine nucleotides

UDP-galactose 4-epimerase contains the coenzyme NAD+ bound tightly at the active site. NAD+ functions as the coenzyme for the interconversion of UDP-galactose and UDP-glucose by reversibly mediating their dehydrogenation to the common intermediate UDP-4-ketohexopyranoside. The epimerase structure and spectrophotometric data indicate that NAD+ may engage in electrostatic interactions with amino acid side chains that may regulate the reactivity of NAD+. In this work, we carried out NMR studies of [nicotinamide-4-13C]NAD+ bound to wild-type epimerase and epimerases mutated at amino acid residues in contact with NAD+. The 4-13C NMR chemical shifts revealed the following: The 4-13C chemical shift in wild-type epimerase is 149.9 ppm; mutation of Ser 124 to Ala changes it slightly by 0.2 ppm to 150.1 ppm; mutation of Tyr 149 to Phe results in a downfield perturbation of 2.7 ppm to 152.6 ppm; and the simultaneous mutation of Ser 124 to Ala and Tyr 149 to Phe also causes a downfield perturbation of 2.8 ppm to 152.7 ppm. Mutation of Lys 153 to Met results in a 13C chemical shift of 150.8 ppm, which is 0.9 ppm downfield from that of wild type and 1.8 ppm upfield from that of Y149F-epimerase. The 13C chemical shifts of nicotinamide C4 of NAD+ in these epimerases are correlated with their respective reactivities with NaBH3CN. In addition, reactivity of NAD+ in wild-type and S124A-epimerases displays pH dependence, with higher rates at lower pH where Tyr 149 in these two enzymes is protonated. The results support an electrostatic model in which repulsion between positively charged Lys 153 and N1 of the nicotinamide ring increases the reactivity of NAD+, while the phenolate of Tyr 149 opposes the positive electrostatic field and attenuates the reactivity of NAD+. Ser 124 has very little effect on the electron distribution within the nicotinamide ring or the reactivity of NAD+. The effects of binding the substrate analogue P1-uridyl-P2-methyl diphosphate (Me-UDP) on the 4-13C chemical shifts are opposite to those induced by the mutations. MeUDP perturbs the 4-13C chemical shift 2.9 ppm downfield in the wild-type and S124A-epimerases but has little or no effect in the cases of Y149F- or K153M-epimerases. The results support the postulate that NAD+ activation induced by uridine nucleotides is brought about by a conformational change of epimerase that repositions Tyr 149 at an increased distance from nicotinamide N1 of NAD+ while maintaining the electrostatic repulsion between Lys 153 and nicotinamide N1 of NAD+.     

High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol.

UDP-galactose 4-epimerase from Escherichia coli catalyzes the interconversion of UDP-glucose and UDP-galactose. In recent years, the enzyme has been the subject of intensive investigation due in part to its ability to facilitate nonstereospecific hydride transfer between beta-NADH and a 4-keto hexopyranose intermediate. The first molecular model of the epimerase from E. coli was solved to 2.5 A resolution with crystals grown in the presence of a substrate analogue, UDP-phenol (Bauer AJ, Rayment I, Frey PA, Holden HM, 1992, Proteins Struct Funct Genet 12:372-381). There were concerns at the time that the inhibitor did not adequately mimic the sugar moiety of a true substrate. Here we describe the high-resolution X-ray crystal structure of the ternary complex of UDP-galactose 4-epimerase with NADH and UDP-phenol. The model was refined to 1.8 A resolution with a final overall R-factor of 18.6%. This high-resolution structural analysis demonstrates that the original concerns were unfounded and that, in fact, UDP-phenol and UDP-glucose bind similarly. The carboxamide groups of the dinucleotides, in both subunits, are displaced significantly from the planes of the nicotinamide rings by hydrogen bonding interactions with Ser 124 and Tyr 149. UDP-galactose 4-epimerase belongs to a family of enzymes known as the short-chain dehydrogenases, which contain a characteristic Tyr-Lys couple thought to be important for catalysis. The epimerase/NADH/UDP-phenol model presented here represents a well-defined ternary complex for this family of proteins and, as such, provides important information regarding the possible role of the Tyr-Lys couple in the reaction mechanism.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: existence of subunit independent functional site

UDP-galactose 4-epimerase from Kluyveromyces fragilis is a stable homodimer of 75 kDa/subunit with non-covalently bound NAD acting as cofactor. Partial proteolysis with trypsin in the presence of 5'-UMP, a strong competitive inhibitor, led to a degraded product which was purified. Results from SDS-PAGE, size-exclusion (SE)-HPLC and ultracentrifugation indicated its monomeric status and size between 43 and 45 kDa. 'Two-step assay' with UDP-glucose dehydrogenase as coupling enzyme in the presence of NAD ensured epimerase activity of the monomer. The possibility of transient dimerization of monomeric epimerase during catalysis was excluded by SE-HPLC in the presence of excess substrate and NAD. This truncated enzyme retained catalytic site related properties like Km for UDP-galactose, 'NADH-like coenzyme fluorescence' and 'reductive inhibition' similar to its dimeric counterpart. Reversible reactivation of the monomer was achieved up to 95% within 3 min from 8 M urea induced unfolded state, indicating that the catalytic site could form independent of its quaternary structure. Equilibrium unfolding between 0 and 8 M urea indicated that the monomer was less stable compared to the dimer. Chemical modification of amino acids and reconstitution with etheno-NAD suggested that the architecture around the catalytic site of the monomer was conserved. Specific modification reagents further confirmed that the cysteine residues required for catalysis and coenzyme fluorophore reside exclusively on a single subunit negating a 'subunit sharing model' of its catalytic site.     

Uridine diphosphate galactose 4-epimerase: nucleotide and 8-anilino-1-naphthalenesulfonate binding properties of the substrate binding site.

Escherichia coli UDP-galactose 4-epimerase in its native form (epimerase.NAD) binds 8-anilino-1-naphthalenesulfonate (ANS) at one tight binding site per dimer with a dissociation constant of 25.9 +/- 2.1 micrometer at pH 8.5 and 27 degrees C. This appears to be the substrate binding site, as indicated by the fact that ANS is a kinetically competitive reversible inhibitor with a Ki of 27.5 micrometer and by the fact that ANS competes with UMP for binding to the enzyme. Upon binding at this site the fluorescence quantum yield of ANS is enhanced 185-fold, and its emission spectrum is blue shifted from a lambdamax of 515 to 470.nm, which suggests that the binding site is shielded from water and probably hydrophobic. Competitive binding experiments with nucleosides and nucleotides indicate that nucleotide binding at this site involves coupled hydrophobic and electrostatic interactions. The reduced form of the enzyme (epimerase.NADH) has no detectable binding affinity for ANS. The marked difference in the affinities of the native and reduced enzymes for ANS is interpreted to be a manifestation of a conformational difference between these enzyme forms.     

Structure and action of urocanase

Urocanase (EC 4.2.1.49) from Pseudomonas putida was crystallized after removing one of the seven free thiol groups. The crystal structure was solved by multiwavelength anomalous diffraction (MAD) using a seleno-methionine derivative and then refined at 1.14 A resolution. The enzyme is a symmetric homodimer of 2 x 557 amino acid residues with tightly bound NAD+ cofactors. Each subunit consists of a typical NAD-binding domain inserted into a larger core domain that forms the dimer interface. The core domain has a novel chain fold and accommodates the substrate urocanate in a surface depression. The NAD domain sits like a lid on the core domain depression and points with the nicotinamide group to the substrate. Substrate, nicotinamide and five water molecules are completely sequestered in a cavity. Most likely, one of these water molecules hydrates the substrate during catalysis. This cavity has to open for substrate passage, which probably means lifting the NAD domain. The observed atomic arrangement at the active center gives rise to a detailed proposal for the catalytic mechanism that is consistent with published chemical data. As expected, the variability of the residues involved is low, as derived from a family of 58 proteins annotated as urocanases in the data banks. However, one well-embedded member of this family showed a significant deviation at the active center indicating an incorrect annotation.     

Localization of translation initiation in the message for E. coli UDP-galactose 4-epimerase: the amino terminal sequence

The amino acid sequence of $\text{E.}$$\text{coli}$ UDP-galactose 4-epimerase has been determined through the amino-terminal 28-amino acid residues using an automated protein sequenator. Alignment of UDP-galactose operon messenger RNA and the amino acid sequence of epimerase demonstrates that the first 26 bases in the mRNA are transcribed but do not take part in translation of epimerase.     

Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation

The binding of Congo red to several purified amyloid-like peptides having a beta-pleated sheet conformation was quantitatively examined. Congo red binds preferentially to the beta-pleated sheet conformation of both insulin fibrils and poly-L-lysine. Congo red does not bind nearly so well to poly-L-serine or polyglycine, despite the fact that these peptides also have a beta-pleated sheet conformation. Binding to insulin fibrils was saturable with an apparent Bmax of 2 moles of Congo red per mole of insulin fibrils and an apparent KD of 1.75 x 10(-7) M. Binding to beta-poly-L-lysine was similar but had a much higher apparent Bmax of 43. Binding of Congo red to beta-poly-L-lysine was pH dependent and appeared to be determined by the number of protonated lysine residues in the 250 amino acid peptide. We present a new hypothesis in which Congo red binds to amyloid-like proteins via bonds between the two negatively charged sulfonic acid groups of Congo red and two positively charged amino acid residues of two separate protein molecules which are properly oriented by virtue of the beta-pleated sheet conformation of the peptide backbone.     

Characterization of hypothetical protein VNG0128C from Halobacterium NRC-1 reveals GALE like activity and its involvement in Leloir pathway of galactose metabolism

VNG0128C, a hypothetical protein from Halobacterium NRC-1, was chosen for detailed insilico and experimental investigations. Computational exercises revealed that VNG0128C functions as NAD⁺ binding protein. The phylogenetic analysis with the homolog sequences of VNG0128C suggested that it could act as UDP-galactose 4-epimerase. Hence, the VNG0128C sequence was modeled using a suitable template and docking studies were performed with NAD and UDP-galactose as ligands. The binding interactions strongly indicate that VNG0128C could plausibly act as UDP-galactose 4-epimerase. In order to validate these insilico results, VNG0128C was cloned in pUC57, subcloned in pET22b⁺, expressed in BL21 cells and purified using nickel affinity chromatography. An assay using blue dextran was performed to confirm the presence of NAD binding domain. To corroborate the epimerase like enzymatic role of the hypothetical protein, i.e. the ability of the enzyme to convert UDP-galactose to UDP-glucose, the conversion of NAD to NADH was measured. The experimental assay significantly correlated with the insilico predictions, indicating that VNG0128C has a NAD⁺ binding domain with epimerase activity. Consequently, its key role in nucleotide-sugar metabolism was thus established. Additionally, the work highlights the need for a methodical characterization of hypothetical proteins (less studied class of biopolymers) to exploit them for relevant applications in the field of biology.     

Presence of bound substrate in lactate dehydrogenase from carp liver

While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45 degrees C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon.     

Crystallization, structure determination and least-squares refinement to 1.75 A resolution of the fatty-acid-binding protein isolated from Manduca sexta L.

The molecular structure of an insect fatty-acid-binding protein isolated from Manduca sexta L. has been determined and refined to a nominal resolution of 1.75 A. Crystals used in the investigation were grown from 1.6 M-ammonium sulfate solutions buffered at pH 4.5 with 50 mM-sodium succinate, and belonged to space group P2(1) with unit cell dimensions of a = 27.5 A, b = 71.0 A, c = 28.7 A and beta = 90.8 degrees. An electron density map, phased with four heavy-atom derivatives and calculated to 2.5 A resolution, allowed for complete tracing of the 131 amino acid residue polypeptide chain. Subsequent least-squares refinement of the model reduced the R-factor from 46.0% to 17.3% using all measured X-ray data from 30.0 A to 1.75 A. Approximately 92% of the amino acid residues fall into classical secondary structural elements including ten strands of anti-parallel beta-pleated sheet, two alpha-helices, one type I turn, three type II turns, four type II' turns and one type III turn. As in other fatty-acid-binding proteins, the overall molecular architecture of the insect molecule consists of ten strands of anti-parallel beta-pleated sheet forming two layers that are nearly orthogonal to one another. A helix-turn-helix motif at the N-terminal portion of the protein flanks one side of the up-and-down beta-barrel. The functional group of the fatty acid is within hydrogen-bonding distance of Gln39, Tyr129, Arg127 and a sulfate molecule, while the aliphatic portion of the ligand is surrounded by hydrophobic amino acid residues lining the beta-barrel. The binding of the carboxylic acid portion of the ligand is very similar to that observed in P2 myelin protein and the murine adipocyte lipid-binding protein, but the positioning of the hydrocarbon tail after approximately C6 is completely different.     

Characterization of Gla(KP), a UDP-galacturonic acid C4-epimerase from Klebsiella pneumoniae with extended substrate specificity

In Escherichia coli and Salmonella enterica, the core oligosaccharide backbone of the lipopolysaccharide is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. In contrast, Klebsiella pneumoniae lacks phosphoryl groups in its core oligosaccharide but instead contains galacturonic acid residues that are proposed to serve a similar function in outer membrane stability. Gla(KP) is a UDP-galacturonic acid C4-epimerase that provides UDP-galacturonic acid for core synthesis, and the enzyme was biochemically characterized because of its potentially important role in outer membrane stability. High-performance anion-exchange chromatography was used to demonstrate the UDP-galacturonic acid C4-epimerase activity of Gla(KP), and capillary electrophoresis was used for activity assays. The reaction equilibrium favors UDP-galacturonic acid over UDP-glucuronic acid in a ratio of 1.4:1, with the K(m) for UDP-glucuronic acid of 13.0 microM. Gla(KP) exists as a dimer in its native form. NAD+/NADH is tightly bound by the enzyme and addition of supplementary NAD+ is not required for activity of the purified enzyme. Divalent cations have an unexpected inhibitory effect on enzyme activity. Gla(KP) was found to have a broad substrate specificity in vitro; it is capable of interconverting UDP-glucose/UDP-galactose and UDP-N-acetylglucosamine/UDP-N-acetylgalactosamine, albeit at much lower activity. The epimerase GalE interconverts UDP-glucose/UDP-galactose. Multicopy plasmid-encoded gla(KP) partially complemented a galE mutation in S. enterica and in K. pneumoniae; however, chromosomal gla(KP) could not substitute for galE in a K. pneumoniae galE mutant in vivo.     

Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-glucose and UDP-galactose during normal galactose metabolism. In humans, deficiencies in this enzyme lead to the complex disorder referred to as epimerase-deficiency galactosemia. Here, we describe the high-resolution X-ray crystallographic structures of human epimerase in the resting state (i.e., with bound NAD(+)) and in a ternary complex with bound NADH and UDP-glucose. Those amino acid side chains responsible for anchoring the NAD(+) to the protein include Asp 33, Asn 37, Asp 66, Tyr 157, and Lys 161. The glucosyl group of the substrate is bound to the protein via the side-chain carboxamide groups of Asn 187 and Asn 207. Additionally, O(gamma) of Ser 132 and O(eta) of Tyr 157 lie within 2.4 and 3.1 A, respectively, of the 4'-hydroxyl group of the sugar. Comparison of the polypeptide chains for the resting enzyme and for the protein with bound NADH and UDP-glucose demonstrates that the major conformational changes which occur upon substrate binding are limited primarily to the regions defined by Glu 199 to Asp 240 and Gly 274 to Tyr 308. Additionally, this investigation reveals for the first time that a conserved tyrosine, namely Tyr 157, is in the proper position to interact directly with the 4'-hydroxyl group of the sugar substrate and to thus serve as the active-site base. A low barrier hydrogen bond between the 4'-hydroxyl group of the sugar and O(gamma) of Ser 132 facilitates proton transfer from the sugar 4'-hydroxyl group to O(eta) of Tyr 157.     

Crystal structure of S-adenosylhomocysteine hydrolase from rat liver.

The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) has been determined at 2.8 A resolution. AdoHcyase from rat liver is a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit is composed of the catalytic domain, the NAD+-binding domain, and the small C-terminal domain. Both catalytic and NAD+-binding domains are folded into an ellipsoid with a typical alpha/beta twisted open sheet structure. The C-terminal section is far from the main body of the subunit and extends into the opposite subunit. An NAD+ molecule binds to the consensus NAD+-binding cleft of the NAD+-binding domain. The peptide folding pattern of the catalytic domain is quite similar to the patterns observed in many methyltransferases. Although the crystal structure does not contain AdoHcy or its analogue, there is a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy molecule can be placed in the catalytic domain. In the structure described here, the catalytic and NAD+-binding domains are quite far apart from each other. Thus, the enzyme appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD+. A catalytic mechanism of AdoHcyase has been proposed on the basis of this crystal structure. Glu155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD+. His54 or Asp130 acts as a general acid-base catalyst, while Cys194 modulates the oxidation state of the bound NAD+. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy molecules can travel freely to and from AdoHcyase and methyltransferases to properly regulate methyltransferase activities. We believe that the crystal structure described here can provide insight into the molecular architecture of this important regulatory enzyme.     

Phenylalanine dehydrogenase from Rhodococcus sp. M4: high-resolution X-ray analyses of inhibitory ternary complexes reveal key features in the oxidative deamination mechanism

The molecular structures of recombinant L-phenylalanine dehydrogenase from Rhodococcus sp. M4 in two different inhibitory ternary complexes have been determined by X-ray crystallographic analyses to high resolution. Both structures show that L-phenylalanine dehydrogenase is a homodimeric enzyme with each monomer composed of distinct globular N- and C-terminal domains separated by a deep cleft containing the active site. The N-terminal domain binds the amino acid substrate and contributes to the interactions at the subunit:subunit interface. The C-terminal domain contains a typical Rossmann fold and orients the dinucleotide. The dimer has overall dimensions of approximately 82 A x 75 A x 75 A, with roughly 50 A separating the two active sites. The structures described here, namely the enzyme.NAD+.phenylpyruvate, and enzyme. NAD+.beta-phenylpropionate species, represent the first models for any amino acid dehydrogenase in a ternary complex. By analysis of the active-site interactions in these models, along with the currently available kinetic data, a detailed chemical mechanism has been proposed. This mechanism differs from those proposed to date in that it accounts for the inability of the amino acid dehydrogenases, in general, to function as hydroxy acid dehydrogenases.     

Evidence that NADP+ is the physiological cofactor of ADP-L-glycero-D-mannoheptose 6-epimerase

ADP-L-glycero-D-mannoheptose 6-epimerase is required for lipopolysaccharide inner core biosynthesis in several genera of Gram-negative bacteria. The enzyme contains both fingerprint sequences Gly-X-Gly-X-X-Gly and Gly-X-X-Gly-X-X-Gly near its N terminus, which is indicative of an ADP binding fold. Previous studies of this ADP-l-glycero-D-mannoheptose 6-epimerase (ADP-hep 6-epimerase) were consistent with an NAD(+) cofactor. However, the crystal structure of this ADP-hep 6-epimerase showed bound NADP (Deacon, A. M., Ni, Y. S., Coleman, W. G., Jr., and Ealick, S. E. (2000) Structure 5, 453-462). In present studies, apo-ADP-hep 6-epimerase was reconstituted with NAD(+), NADP(+), and FAD. In this report we provide data that shows NAD(+) and NADP(+) both restored enzymatic activity, but FAD could not. Furthermore, ADP-hep 6-epimerase exhibited a preference for binding of NADP(+) over NAD(+). The K(d) value for NADP(+) was 26 microm whereas that for NAD(+) was 45 microm. Ultraviolet circular dichroism spectra showed that apo-ADP-hep 6-epimerase reconstituted with NADP(+) had more secondary structure than apo-ADP-hep 6-epimerase reconstituted with NAD(+). Perchloric acid extracts of the purified enzyme were assayed with NAD(+)-specific alcohol dehydrogenase and NADP(+)-specific isocitric dehydrogenase. A sample of the same perchloric acid extract was analyzed in chromatographic studies, which demonstrated that ADP-hep 6-epimerase binds NADP(+) in vivo. A structural comparison of ADP-hep 6-epimerase with UDP-galactose 4-epimerase, which utilizes an NAD(+) cofactor, has identified the regions of ADP-hep 6-epimerase, which defines its specificity for NADP(+).     

The 2 A resolution structure of the sulfate-binding protein involved in active transport in Salmonella typhimurium

The crystal structure of the liganded form of the sulfate-binding protein, an initial receptor for active transport of sulfate in Salmonella typhimurium, has been solved and refined at 2.0 A resolution (1 A = 0.1 nm). The final model, which consists of 2422 non-hydrogen atoms, one sulfate substrate and 143 water molecules, yields a crystallographic R-factor of 14.0% for 16,959 reflections between 8 and 2 A. The structure deviates from ideal bond lengths and angle distances by 0.015 A and 0.037 A, respectively. The protein is ellipsoid with overall dimensions of 35 A x 35 A x 65 A and consists of two similar globular domains. The two domains are linked by three distinct peptide segments, which though widely separated in the amino acid sequence, are in close proximity in the tertiary structure. As these connecting segments are located near the periphery of the molecule, they further serve as the base or a "boundary" of the deep cleft formed between the two domains. Despite the unusual interdomain connectivity, both domains have similar supersecondary structure consisting of a central five-stranded beta-pleated sheet sandwiched by alpha-helices on either side. The arrangement of the two domains gives rise to the ellipsoidal shape and to the cleft between the two domains wherein the sulfate substrate is found and completely engulfed. A discovery of considerable importance is that the sulfate substrate is tightly held in place primarily by seven hydrogen bonds, five of which are donated by main-chain peptide NH groups, another by a serine hydroxyl and the last by the indole NH moiety of a tryptophan side-chain; there are no positively charged residues, nor cations, nor water molecules within van der Waals' distance to the sulfate dianion. All the main-chain peptide units associated with the sulfate are in turn linked (via the peptide CO group) to arrays of hydrogen bonds. Three of these arrays are composed of alternating peptide units and hydrogen bonds within the solvent-exposed part of three alpha-helices and two are linked to a histidine and an arginine residue. The sulfate-binding protein bears strong similarity to the structures of four other periplasmic binding proteins solved in our laboratory which are specific for L-arabinose, D-galactose/D-glucose, leucine/isoleucine/valine and leucine. The similarity includes the ellipsoidal shape and the two globular domain structures, each domain consisting of a central beta-pleated sheet flanked by alpha-helices.(ABSTRACT TRUNCATED AT 400 WORDS)     

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase has been determined at 2.7 A resolution. Its first 120 amino acids form a central five-stranded, beta-pleated sheet surrounded by five alpha helices. In one of the four dyad-related dimers, the two active site Ser-10 residues are 19 A apart, perhaps close enough to contact and become covalently linked to the DNA at the recombination site. This dimer also forms the only closely packed tetramer found in the crystal. The subunit interface at a second dyad-related dimer is more extensive and more highly conserved among the homologous recombinases; however, its active site Ser-10 residues are more than 30 A apart. Side chains, identified by mutations that eliminate catalysis but not DNA binding, are located on the subunit surface near the active site serine and at the interface between a third dyad-related pair of subunits of the tetramer.