EXAFS studies of the zinc sites of UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC)

Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the structure of the Zn(II) sites in UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) from Aquifex aeolicus and Pseudomonas aeruginosa. The active site Zn(II) is four coordinate, with exclusively low-Z (nitrogen and oxygen) ligation in both enzymes. The amplitude of the outer-shell scattering from the histidine ligands is best fit using two histidine ligands, suggesting a ZnO(2)(His)(2) site, where O most likely represents a conserved aspartate and a solvent molecule. The same structure was found for Co(II)-substituted A. aeolicus LpxC, although in this case it is possible that the coordination sphere may expand to include a fifth low-Z ligand. EXAFS data were also measured for the Escherichia coli LpxC enzyme. When a single Co(II) is substituted for Zn(II) in the active site of E. coli LpxC, EXAFS data show the same ligand environment as is found for the P. aeruginosa and A. aeolicus enzymes. However, the EXAFS data for E. coli LpxC with two zinc ions bound per protein, with the second Zn(II) acting as an inhibitory metal, demonstrates that the inhibitory metal is bound to at least two high-Z (sulfur, presumably thiolate, or chlorine) ligands. Results of the outer-shell scattering analysis, combined with previous studies of the LpxC enzyme, indicate a novel zinc binding motif not found in any previously studied zinc metalloproteins.     

Origins of blood acetate in the rat.

1. Fluorimetric techniques were used to characterize the environment of tryptophan residues in thermolysin and apo-thermolysin. The apo-thermolysin was obtained by dissolving the enzyme in the presence of 10mm-EDTA, which removed the functional Zn(2+) ion and the four Ca(2+) ions/molecule from the enzyme. 2. At 25 degrees C in aqueous solution the fluorescence-emission spectrum of the native holoenzyme, on excitation at 290nm, was essentially characteristic of tryptophan, with an emission maximum at 333nm. The emission maximum of the apoenzyme is red-shifted to 338nm and the relative intensity of fluorescence is decreased by 10%, both effects indicating some unfolding of the protein molecule, with the indole groups being transferred to a more hydrophilic environment. 3. Fluorescence quenching studies using KI, N'-methylnicotinamide hydrochloride and acrylamide indicated a more open structure in the apoenzyme, with the tryptophan residues located in a negatively charged environment. 4. The thermal properties of the apoenzyme, as monitored by fluorescence-emission measurements, are dramatically changed with respect to the native holoenzyme. In fact, whereas the native enzyme is heat-stable up to about 80 degrees C, for the apoenzyme a thermal transition is observed near 48 degrees C. The apoenzyme is also unstable to the action of unfolding agents such as urea and guanidinium chloride, much as for other globular proteins from mesophilic organisms. 5. The functional Zn(2+) ion does not contribute noticeably to the stability of thermolysin. 6. It is concluded that a major role in the structural stability of thermolysin is played by the Ca(2+) ions, which have a bridging function within this disulphide-free protein molecule.     

Why does Escherichia coli grow more slowly on glucosamine than on N-acetylglucosamine? Effects of enzyme levels and allosteric activation of GlcN6P deaminase (NagB) on growth rates

Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.     

The crystal and solution studies of glucosamine-6-phosphate synthase from Candida albicans

Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity and details of the isomerase active site.     

Probing the role of metal ions in the catalysis of Helicobacter pylori 3-deoxy-D-manno-octulosonate-8-phosphate synthase using a transient kinetic analysis

3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the net condensation of phosphoenolpyruvate and d-arabinose 5-phosphate to form KDO8P and inorganic phosphate (Pi). Two classes of KDO8P synthases have been identified. The Class I KDO8P synthases (e.g. Escherchia coli KDO8P synthase) catalyze the condensation reaction in a metal-independent fashion, whereas the Class II enzymes (e.g. Aquifex aeolicus) require metal ions for catalysis. Helicobacter pylori (H. pylori) KDO8P synthase, a Zn2+-dependent metalloenzyme, has recently been found to be a Class II enzyme and has a high degree of clinical significance since it is an attractive molecular target for the design of novel antibiotic therapy. Although the presence of a divalent metal ion in Class II KDO8P synthases is essential for catalysis, there is a paucity of mechanistic information on the role of the metal ions and functional differences as compared with Class I enzymes. Using H. pylori KDO8P synthase as a prototypical Class II enzyme, a steady-state and transient kinetic approach was undertaken to understand the role of the metal ion in catalysis and define the kinetic reaction pathway. Metal reconstitution experiments examining the reaction kinetics using Zn2+, Cd2+, Cu2+, Co2+, Mn2+, and Ni2+ yielded surprising results in that the Cd2+ enzyme has the greatest activity. Unlike Class-I KDO8P synthases, the Class II metallo-KDO8P synthases containing Zn2+, Cd2+, Cu2+, and Co2+ show cooperativity. This study presents the first detailed kinetic characterization of a metal-dependent Class II KDO8P synthase and offers mechanistic insight for how the divalent metal ions modulate catalysis through effects on chemistry as well as quaternary protein structure.     

Effect of Zn(II) and Mg(II) on phosphohydrolytic activity of rat matrix-induced alkaline phosphatase

Rat matrix-induced alkaline phosphatase is an enzyme which requires magnesium and zinc ions for its maximal activity. Two Zn(II) ions and one Mg(II) ion are bound to each subunit of native dimeric enzyme. The presence of magnesium ion (10-100 microM) or zinc ion (7-20 nM) alone is sufficient to stimulate apoenzyme activity. However maximal activity (264 U/mg) requires the presence of both ions. Binding of Zn(II) ions to the Mg(II) binding site causes a strong inhibition of the apoenzyme while the binding of Mg(II) on Zn(II) binding site is not sufficient to stimulate PNPPase activity of the apoenzyme. Binding of both ions to the enzyme molecule did not change the apparent dissociation constant for PNPP hydrolysis.     

Allosteric kinetics of the isoform 1 of human glucosamine-6-phosphate deaminase

The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.6). Isoform 1 has been purified from several animal sources and the crystallographic structure of the human recombinant enzyme was solved at 1.75? resolution (PDB ID: 1NE7). In spite of their great structural similarity, human and Escherichia coli GNPDAs show marked differences in their allosteric kinetics. The allosteric site ligand, N-acetylglucosamine 6-phosphate (GlcNAc6P), which is an activator of the K-type of E. coli GNPDA has an unusual mixed allosteric effect on hGNPDA1, behaving as a V activator and a K inhibitor (antiergistic or crossed mixed K(-)V(+) effect). In the absence of GlcNAc6P, the apparent k(cat) of the enzyme is so low, that GlcNAc6P behaves as an essential activator. Additionally, substrate inhibition, dependent on GlcNAc6P concentration, is observed. All these kinetic properties can be well described within the framework of the Monod allosteric model with some additional postulates. These unusual kinetic properties suggest that hGNPDA1 could be important for the maintenance of an adequate level of the pool of the UDP-GlcNAc6P, the N-acetylglucosylaminyl donor for many reactions in the cell. In this research we have also explored the possible functional significance of the C-terminal extension of hGNPDA1 enzyme, which is not present in isoform 2, by constructing and studying two mutants truncated at positions 268 and 275.     

Acceptor substrate binding revealed by crystal structure of human glucosamine-6-phosphate N-acetyltransferase 1

Glucosamine-6-phosphate (GlcN6P) N-acetyltransferase 1 (GNA1) is a key enzyme in the pathway toward biosynthesis of UDP-N-acetylglucosamine, an important donor substrate for N-linked glycosylation. GNA1 catalyzes the formation of N-acetylglucosamine-6-phosphate (GlcNAc6P) from acetyl-CoA (AcCoA) and the acceptor substrate GlcN6P. Here, we report crystal structures of human GNA1, including apo GNA1, the GNA1-GlcN6P complex and an E156A mutant. Our work showed that GlcN6P binds to GNA1 without the help of AcCoA binding. Structural analyses and mutagenesis studies have shed lights on the charge distribution in the GlcN6P binding pocket, and an important role for Glu156 in the substrate binding. Hence, these findings have broadened our knowledge of structural features required for the substrate affinity of GNA1. STRUCTURED SUMMARY:     

Crystallographic and X-ray absorption spectroscopic characterization of Helicobacter pylori UreE bound to Ni²⁺ and Zn²⁺ reveals a role for the disordered C-terminal arm in metal trafficking

The survival and growth of the pathogen Helicobacter pylori in the gastric acidic environment is ensured by the activity of urease, an enzyme containing two essential Ni2? ions in the active site. The metallo-chaperone UreE facilitates in vivo Ni2? insertion into the apoenzyme. Crystals of apo-HpUreE (H. pylori UreE) and its Ni?- and Zn?-bound forms were obtained from protein solutions in the absence and presence of the metal ions. The crystal structures of the homodimeric protein, determined at 2.00 ? (apo), 1.59 ? (Ni2?) and 2.52 ? (Zn2?) resolution, show the conserved proximal and solvent-exposed His1?2 residues from two adjacent monomers invariably involved in metal binding. The C-terminal regions of the apoprotein are disordered in the crystal, but acquire significant ordering in the presence of the metal ions due to the binding of His1?2. The analysis of X-ray absorption spectral data obtained using solutions of Ni2?- and Zn2?-bound HpUreE provided accurate information of the metal-ion environment in the absence of solid-state effects. These results reveal the role of the histidine residues at the protein C-terminus in metal-ion binding, and the mutual influence of protein framework and metal-ion stereo-electronic properties in establishing co-ordination number and geometry leading to metal selectivity.     

Purification and Characterization of N-Acetylglucosamine-6-phosphate Deacetylase from a Psychrotrophic Marine Bacterium, Alteromonas Species

A psychrotrophic bacterium, strain Mct-9, which produced an N-acetylglucosamine-6-phosphate deacetylase, was isolated from a deep-seawater sample in the Mariana Trough. The Mct-9 strain was identified as Alteromonas sp. The native enzyme had a molecular mass of 164,000 Da, and was predicted to be composed of four identical subunits with molecular masses of 41,000 Da. The purified enzyme hydrolyzed N-acetylglucosamine (GlcNAc), GlcNAc-6-phosphate, and GlcNAc-6-sulfate. Considering the low K _m and high k _cat /K _m for GlcNAc-6-phosphate, it probably acts as a GlcNAc-6-phosphate deacetylase in vivo. The enzyme was functional in the temperature range of 5° to 70°C and displayed optimal activity at 55°C. The optimal temperature was higher than that of the deacetylase from the mesophilic bacterium Vibrio cholerae non-O1. The characteristics of the GlcNAc-6-phosphate deacetylase from Alteromonas sp. are unique among psychrotrophs and psychrophiles, whose intracellular enzymes are mostly thermolabile. Received May 6, 1999; accepted August 16, 1999.     

Zinc stoichiometry in Escherichia coli alkaline phosphatase. Studies by 31P NMR and ion-exchange chromatography.

31P nuclear magnetic resonance spectra and enzymatic activities are compared for alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) species with different zinc contents. The enzyme containing two Zn2+ per protein dimer exists in two forms; one, prepared by dialysis of native enzyme, has full enzymatic activity and a 31P magnetic resonance spectrum similar to but distinguishable from that of the native enzyme containing four or more Zn2+. The other form, prepared by restoring two Zn2+ to apoenzyme, has low enzymatic activity and a 31P magnetic resonance spectrum that indicates stoichiometric binding of phosphate, but otherwise altered properties. Reconstituted enzyme with four Zn2+ is similar to but distinguishable from native enzyme with four Zn2+. Chromatography on DEAE-cellulose can separate apoenzyme and enzyme containing two Zn2+ and suggests that the binding of a pair of Zn2+ is cooperative.     

Structure of Escherichia coli uracil DNA glycosylase and its complexes with nonhydrolyzable substrate analogues in solution observed by synchrotron small-angle X-ray scattering

The structure of native and modified uracil DNA glycosylase from E. coli in solution was studied by synchrotron small-angle X-ray scattering. The modified enzyme (6His-uracyl DNA glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence amino acid residues including a block of six His residues. It was found that the conformations of these enzymes in solution at moderate ionic strength (60 mM NaCI) substantially differ in spite of minimal differences in the amino acid sequences and functional activity. The structure of native uracil DNA glycosylase in solution is close to that in crystal, showing a tendency for association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes a partial dissociation of associates and a compactization of protein structure. At the same time, 6His-uracyl DNA glycosylase has a compact structure essentially different from the crystal one. A decrease in the ionic strength of solution results in a partial disruption of compact structure of the modified protein, without changes in its functional activity.     

Resonant X-Ray Scattering and Absorption for the Global and Local Structures of Cu-modified Metallothioneins in Solution

With Cd and Zn metal ions removed from the native rabbit-liver metallothionein upon unfolding, Cu-modified metallothioneins (Cu-MTs) were obtained during refolding in solutions containing Cu^I or Cu^II ions. X-ray absorption near-edge spectroscopic results confirm the respectively assigned oxidation states of the copper ions in Cu^I-MT and Cu^II-MT. Global and local structures of the Cu-MTs were subsequently characterized by anomalous small-angle x-ray scattering (ASAXS) and extended x-ray absorption fine structure. Energy-dependent ASAXS results indicate that the morphology of Cu^II-MT resembles that of the native MT, whereas Cu^I-MT forms oligomers with a higher copper content. Both dummy-residue simulation and model-shape fitting of the ASAXS data reveal consistently rodlike morphology for Cu^II-MT. Clearly identified Cu-S, Cu-O, and Cu-Cu contributions in the extended x-ray absorption fine structure analysis indicate that both Cu^I and Cu^II ions are bonded with O and S atoms of nearby amino acids in a four-coordination environment, forming metal clusters smaller than metal thiolate clusters in the native MT. It is demonstrated that a combination of resonant x-ray scattering and x-ray absorption can be particularly useful in revealing complementary global and local structures of metalloproteins due to the atom specific characteristics of the two techniques.     

Zinc (II) coordination in Escherichia coli DNA topoisomerase I is required for cleavable complex formation with DNA.

Escherichia coli DNA topoisomerase I catalyzes relaxation of negatively supercoiled DNA. The reaction proceeds through a covalent intermediate, the cleavable complex, in which the DNA is cleaved and the enzyme is linked to the DNA via a phosphotyrosine linkage. Each molecule of E. coli DNA topoisomerase I has been shown to have three tightly bound zinc(II) ions required for relaxation activity (Tse-Dinh, Y.-C., and Beran-Steed, R.K. (1988) J. Biol. Chem. 263, 15857-15859). It is shown here that Cd(II) could replace Zn(II) in reconstitution of active enzyme from apoprotein. The role of metal was analyzed by studying the partial reactions. The apoenzyme was deficient in sodium dodecyl sulfate-induced cleavage of supercoiled PM2 phage DNA. Formation of covalent complex with linear single-stranded DNA was also reduced in the absence of metal. However, the cleavage of small oligonucleotide was not affected, and the apoenzyme could religate the covalently bound oligonucleotide to another DNA molecule. Assay of noncovalent complex formation by retention of 5'-labeled DNA on filters showed that the apoenzyme was not inhibited in noncovalent binding to DNA. It is proposed that zinc(II) coordination in E. coli DNA topoisomerase I is required for the transition of the noncovalent complex with DNA to the cleavable state.     

X-ray structure of putative acyl-ACP desaturase DesA2 from Mycobacterium tuberculosis H37Rv

Genome sequencing showed that two proteins in Mycobacterium tuberculosis H37Rv contain the metal binding motif (D/E)X(2)HX(approximately 100)(D/E)X(2)H characteristic of the soluble diiron enzyme superfamily. These putative acyl-ACP desaturase genes desA1 and desA2 were cloned from genomic DNA and expressed in Escherichia coli BL21(DE3). DesA1 was found to be insoluble, but in contrast, DesA2 was a soluble protein amenable to biophysical characterization. Here, we report the 2.0 A resolution X-ray structure of DesA2 determined by multiple anomalous dispersion (MAD) phasing from a Se-met derivative and refinement against diffraction data obtained on the native protein. The X-ray structure shows that DesA2 is a homodimeric protein with a four-helix bundle core flanked by five additional helices that overlay with 192 structurally equivalent amino acids in the structure of stearoyl-ACP Delta9 desaturase from castor plant with an rms difference 1.42 A. In the DesA2 crystals, one metal (likely Mn from the crystallization buffer) was bound in high occupancy at the B-site of the conserved metal binding motif, while the A-site was not occupied by a metal ion. Instead, the amino group of Lys-76 occupied this position. The relationships between DesA2 and known diiron enzymes are discussed.     

Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase

A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.     

Crystal structure of phenylalanine-regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

BACKGROUND: In microorganisms and plants the first step in the common pathway leading to the biosynthesis of aromatic compounds is the stereospecific condensation of phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate (E4P) giving rise to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP). This reaction is catalyzed by DAHP synthase (DAHPS), a metal-activated enzyme, which in microorganisms is the target for negative-feedback regulation by pathway intermediates or by end products. In Escherichia coli there are three DAHPS isoforms, each specifically inhibited by one of the three aromatic amino acids. RESULTS: The crystal structure of the phenylalanine-regulated form of DAHPS complexed with PEP and Pb2+ (DAHPS(Phe)-PEP-Pb) was determined by multiple wavelength anomalous dispersion phasing utilizing the anomalous scattering of Pb2+. The tetramer consists of two tight dimers. The monomers of the tight dimer are coupled by extensive interactions including a pair of three-stranded, intersubunit beta sheets. The monomer (350 residues) is a (beta/alpha)8 barrel with several additional beta strands and alpha helices. The PEP and Pb2+ are at the C-ends of the beta strands of the barrel, as is SO4(2-), inferred to occupy the position of the phosphate of E4P. Mutations that reduce feedback inhibition cluster about a cavity near the twofold axis of the tight dimer and are centered approximately 15 A from the active site, indicating the location of a separate regulatory site. CONCLUSIONS: The crystal structure of DAHPS(Phe)-PEP-Pb reveals the active site of this key enzyme of aromatic biosynthesis and indicates the probable site of inhibitor binding. This is the first reported structure of a DAHPS; the structure of its two paralogs and of a variety of orthologs should now be readily determined by molecular replacement.     

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.     

Crystal structures of human glycerol 3-phosphate dehydrogenase 1 (GPD1)

Homo sapiens L-alpha-glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes the reversible biological conversion of dihydroxyacetone (DHAP) to glycerol-3-phosphate. The GPD1 protein was expressed in Escherichia coli, and purified as a fusion protein with glutathione S-transferase. Here we report the apoenzyme structure of GPD1 determined by multiwavelength anomalous diffraction phasing, and other complex structures with small molecules (NAD+ and DHAP) by the molecular replacement method. This enzyme structure is organized into two distinct domains, the N-terminal eight-stranded beta-sheet sandwich domain and the C-terminal helical substrate-binding domain. An electrophilic catalytic mechanism by the epsilon-NH3+ group of Lys204 is proposed on the basis of the structural analyses. In addition, the inhibitory effects of zinc and sulfate on GPDHs are assayed and discussed.