The structure of holo and metal-deficient wild-type human Cu,Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis

Cu, Zn superoxide dismutase (SOD1) forms a crucial component of the cellular defence against oxidative stress. Zn-deficient wild-type and mutant human SOD1 have been implicated in the disease familial amyotrophic lateral sclerosis (FALS). We present here the crystal structures of holo and metal-deficient (apo) wild-type protein at 1.8A resolution. The P21 wild-type holo enzyme structure has nine independently refined dimers and these combine to form a "trimer of dimers" packing motif in each asymmetric unit. There is no significant asymmetry between the monomers in these dimers, in contrast to the subunit structures of the FALS G37R mutant of human SOD1 and in bovine Cu,Zn SOD. Metal-deficient apo SOD1 crystallizes with two dimers in the asymmetric unit and shows changes in the metal-binding sites and disorder in the Zn binding and electrostatic loops of one dimer, which is devoid of metals. The second dimer lacks Cu but has approximately 20% occupancy of the Zn site and remains structurally similar to wild-type SOD1. The apo protein forms a continuous, extended arrangement of beta-barrels stacked up along the short crystallographic b-axis, while perpendicular to this axis, the constituent beta-strands form a zig-zag array of filaments, the overall arrangement of which has a similarity to the common structure associated with amyloid-like fibrils.     

Structures of D-xylose isomerase from Arthrobacter strain B3728 containing the inhibitors xylitol and D-sorbitol at 2.5 A and 2.3 A resolution, respectively

The structures of D-xylose isomerase from Arthrobacter strain B3728 containing the polyol inhibitors xylitol and D-sorbitol have been solved at 2.5 A and 2.3 A, respectively. The structures have been refined using restrained least-squares refinement methods. The final crystallographic R-factors for the D-sorbitol (xylitol) bound molecules, for 43,615 (32,989) reflections are 15.6 (14.7). The molecule is a tetramer and the asymmetric unit of the crystal contains a dimer, the final model of which, incorporates a total of 6086 unique protein, inhibitor and magnesium atoms together with 535 bound solvent molecules. Each subunit of the enzyme contains two domains: the main domain is a parallel-stranded alpha-beta barrel, which has been reported in 14 other enzymes. The C-terminal domain is a loop structure consisting of five helical segments and is involved in intermolecular contacts between subunits that make up the tetramer. The structures have been analysed with respect to molecular symmetry, intersubunit contacts, inhibitor binding and active site geometry. The refined model shows the two independent subunits to be similar apart from local deviations due to solvent contacts in the solvent-exposed helices. The enzyme is dependent on a divalent cation for catalytic activity. Two metal ions are required per monomer, and the high-affinity magnesium(II) site has been identified from the structural results presented here. The metal ion is complexed, at the high-affinity site, by four carboxylate side-chains of the conserved residues, Glu180, Glu216, Asp244 and Asp292. The inhibitor polyols are bound in the active site in an extended open chain conformation and complete an octahedral co-ordination shell for the magnesium cation via their oxygen atoms O-2 and O-4. The active site lies in a deep pocket near the C-terminal ends of the beta-strands of the barrel domain and includes residues from a second subunit. The tetrameric molecule can be considered to be a dimer of "active" dimers, the active sites being composed of residues from both subunits. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. One of these, between Asp23 and Arg139, appears to play a key role in stabilizing the active dimer and is conserved in the known sequences of this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: evidence of conformational heterogeneity

The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined "open" and "closed" conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5A resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution.     

The structural basis of substrate activation in yeast pyruvate decarboxylase. A crystallographic and kinetic study.

The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered. In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer-dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder-order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed.     

The projection structure of EmrE, a proton-linked multidrug transporter from Escherichia coli, at 7 A resolution

EmrE belongs to a family of eubacterial multidrug transporters that confer resistance to a wide variety of toxins by coupling the influx of protons to toxin extrusion. EmrE was purified and crystallized in two dimensions by reconstitution with dimyristoylphosphatidylcholine into lipid bilayers. Images of frozen hydrated crystals were collected by cryo-electron microscopy and a projection structure of EmrE was calculated to 7 A resolution. The projection map shows an asymmetric EmrE dimer with overall dimensions approximately 31 x 40 A, comprising an arc of highly tilted helices separating two helices nearly perpendicular to the membrane from another two helices, one tilted and the other nearly perpendicular. There is no obvious 2-fold symmetry axis perpendicular to the membrane within the dimer, suggesting that the monomers may have different structures in the functional unit.     

Multiple active site conformations revealed by distant site mutation in ornithine decarboxylase

Ornithine decarboxylase (ODC) is an obligate homodimer that catalyzes the pyridoxal 5'-phosphate-dependent decarboxylation of l-ornithine to putrescine, a vital step in polyamine biosynthesis. A previous mutagenic analysis of the ODC dimer interface identified several residues that were distant from the active site yet had a greater impact on catalytic activity than on dimer stability [Myers, D. P., et al. (2001) Biochemistry 40, 13230-13236]. To better understand the basis of this phenomenon, the structure of the Trypanosoma brucei ODC mutant K294A was determined to 2.15 A resolution in complex with the substrate analogue d-ornithine. This residue is distant from the reactive center (>10 A from the PLP Schiff base), and its mutation reduced catalytic efficiency by 3 kcal/mol. The X-ray structure demonstrates that the mutation increases the disorder of residues Leu-166-Ala-172 (Lys-169 loop), which normally form interactions with Lys-294 across the dimer interface. In turn, the Lys-169 loop forms interactions with the active site, suggesting that the reduced catalytic efficiency is mediated by the decreased stability of this loop. The extent of disorder varies in the four Lys-169 loops in the asymmetric unit, suggesting that the mutation has led to an increase in the population of inactive conformations. The structure also reveals that the mutation has affected the nature of the ligand-bound species. Each of the four active sites contains unusual ligands. The electron density suggests one active site contains a gem-diamine intermediate with d-ornithine; the second has density consistent with a tetrahedral adduct with glycine, and the remaining two contain tetrahedral adducts of PLP, Lys-69, and water (or hydroxide). These data also suggest that the structure is less constrained in the mutant enzyme. The observation of a gem-diamine intermediate provides insight into the conformational changes that occur during the ODC catalytic cycle.     

Refined structure of chicken skeletal muscle troponin C in the two-calcium state at 2-A resolution

The structure of troponin C has been refined at 2A resolution to an R value of 0.172 using a total of 8,100 reflections. Troponin C has an unusual dumbbell shape with only the two C-domain high affinity sites III and IV occupied with metals, while the pair of N-domain low affinity sites I and II are devoid of metals. The coordination of the Ca2+ approaches seven with the last glutamic acid residue in each site forming an asymmetric bidentate ligand. The flanking helices in the metal-bound EF hands are in similar orientation (both 113 degrees) while in the apo sites they are more obtuse (134 and 149 degrees). The EF hands of holo sites III and IV are similar while the apo sites I and II are less similar (rms for backbone atoms, 0.78 and 1.44). The half-loops of the 12-residue holo and apo sites show better agreement than the full loops themselves, suggesting a hinge motion at the midpoint of the loops. The long central helix is stabilized by electrostatic interactions and salt bridges between charged side chains spaced at 3 or 4 residues along the helix. A cluster of water molecules encircle the long helix and hydrogen bond to the backbone carbonyls. At the beginning of the B-helix, a water molecule is interposed at each of two consecutive backbone NH...OC hydrogen bonds. The terminal pair of helices A/D (apo) match with E/H (holo), and the internal pair of helices B/C (apo) match with F/G (holo). Thus, muscle contraction may be triggered by Ca2+ binding to loops I and II which results in a concerted rearrangement of residues in the loops, including the essential Gly at position 6 in each loop. This rearrangement than causes a reorientation of helices B and C along with the BC linker.     

Crystal structure and dimerization equilibria of PcoC, a methionine-rich copper resistance protein from Escherichia coli

PcoC is a soluble periplasmic protein encoded by the plasmid-born pco copper resistance operon of Escherichia coli. Like PcoA, a multicopper oxidase encoded in the same locus and its chromosomal homolog CueO, PcoC contains unusual methionine rich sequences. Although essential for copper resistance, the functions of PcoC, PcoA, and their conserved methionine-rich sequences are not known. Similar methionine motifs observed in eukaryotic copper transporters have been proposed to bind copper, but there are no precedents for such metal binding sites in structurally characterized proteins. The high-resolution structures of apo PcoC, determined for both the native and selenomethionine-containing proteins, reveal a seven-stranded beta barrel with the methionines unexpectedly housed on a solvent-exposed loop. Several potential metal-binding sites can be discerned by comparing the structures to spectroscopic data reported for copper-loaded PcoC. In the native structure, the methionine loop interacts with the same loop on a second molecule in the asymmetric unit. In the selenomethionine structure, the methionine loops are more exposed, forming hydrophobic patches on the protein surface. These two arrangements suggest that the methionine motifs might function in protein-protein interactions between PcoC molecules or with other methionine-rich proteins such as PcoA. Analytical ultracentrifugation data indicate that a weak monomer-dimer equilibrium exists in solution for the apo protein. Dimerization is significantly enhanced upon binding Cu(I) with a measured delta(deltaG degrees )相似文献   

Crystal structure of ATP phosphoribosyltransferase from Mycobacterium tuberculosis

The N-1-(5'-phosphoribosyl)-ATP transferase catalyzes the first step of the histidine biosynthetic pathway and is regulated by a feedback mechanism by the product histidine. The crystal structures of the N-1-(5'-phosphoribosyl)-ATP transferase from Mycobacterium tuberculosis in complex with inhibitor histidine and AMP has been determined to 1.8 A resolution and without ligands to 2.7 A resolution. The active enzyme exists primarily as a dimer, and the histidine-inhibited form is a hexamer. The structure represents a new fold for a phosphoribosyltransferase, consisting of three continuous domains. The inhibitor AMP binds in the active site cavity formed between the two catalytic domains. A model for the mechanism of allosteric inhibition has been derived from conformational differences between the AMP:His-bound and apo structures.     

Crystal structure of a ternary complex of the alcohol dehydrogenase from Sulfolobus solfataricus

The crystal structure of a ternary complex of the alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH) has been determined at 2.3 A. The asymmetric unit contains a dimer with a NADH and a 2-ethoxyethanol molecule bound to each subunit. The comparison with the apo structure of the enzyme reveals that this medium chain ADH undergoes a substantial conformational change in the apo-holo transition, accompanied by loop movements at the domain interface. The extent of domain closure is similar to that observed for the classical horse liver ADH, although some differences are found which can be related to the different oligomeric states of the enzymes. Compared to its apo form, the SsADH ternary complex shows a change in the ligation state of the active site zinc ion which is no longer bound to Glu69, providing additional evidence of the dynamic role played by the conserved glutamate residue in ADHs. In addition, the structure presented here allows the identification of the substrate site and hence of the residues that are important in the binding of both the substrate and the coenzyme.     

Structure of Plasmodium falciparum triose-phosphate isomerase-2-phosphoglycerate complex at 1.1-A resolution

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-A resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208-211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue.     

Crystal structure of E.coli alcohol dehydrogenase YqhD: evidence of a covalently modified NADP coenzyme

In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame (ORF) products of unknown function, we have determined the structure of YqhD at 2.0A resolution using the single wavelength anomalous diffraction method at the Pt edge. The crystal structure of YqhD reveals that it is an NADP-dependent dehydrogenase, a result confirmed by activity measurements with several alcohols. The current interpretation of our findings is that YqhD is an alcohol dehydrogenase (ADH) with preference for alcohols longer than C(3). YqhD is a dimer of 2x387 residues, each monomer being composed of two domains, a Rossmann-type fold and an alpha-helical domain. The crystals contain two dimers in the asymmetric unit. While one of the dimers contains a cofactor in both subunits, only one of the subunits in the second dimer contains it, making it possible to compare bound and unbound active sites. The active site contains a Zn atom, as verified by EXAFS on the crystals. The electron density maps of NADP revealed modifications of the nicotinamide ring by oxygen atoms at positions 5 and 6. Further analysis by electrospray mass spectrometry and comparison with the mass spectra of NADP and NADPH revealed the nature of the modification and the incorporation of two hydroxyl moieties at the 5 and 6 position in the nicotinamide ring, yielding NADPH(OH)(2). These modifications might be due to oxygen stress on an enzyme, which would functionally work under anaerobic conditions.     

The crystal structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase.

In animals, glycogen phosphorylase (GP) exists in an inactive (T state) and an active (R state) equilibrium that can be altered by allosteric effectors or covalent modification. In Escherichia coli, the activity of maltodextrin phosphorylase (MalP) is controlled by induction at the level of gene expression, and the enzyme exhibits no regulatory properties. We report the crystal structure of E. coli maltodextrin phosphorylase refined to 2.4 A resolution. The molecule consists of a dimer with 796 amino acids per monomer, with 46% sequence identity to the mammalian enzyme. The overall structure of MalP shows a similar fold to GP and the catalytic sites are highly conserved. However, the relative orientation of the two subunits in E. coli MalP is different from both the T and R state GP structures, and there are significant changes at the subunit-subunit interfaces. The sequence changes result in loss of each of the control sites present in rabbit muscle GP. As a result of the changes at the subunit interface, the 280s loop, which in T state GP acts as a gate to control access to the catalytic site, is held in an open conformation in MalP. The open access to the conserved catalytic site provides an explanation for the activity without control in this basic archetype of a phosphorylase.     

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of ∼3.5 Å in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action.     

The crystal structures of recombinant glycosylated human renin alone and in complex with a transition state analog inhibitor

Recombinant human glycosylated renin has been crystallized in complex with CGP 38'560, a transition state analog inhibitor (IC50 = 2 x 10(-9) M), in a tetragonal crystal form. The structure has been determined to a resolution of 2.4 A and refined to a crystallographic Rfactor of 17.6%. It reveals the conformation of the inhibitor as well as its interactions with the enzyme active site. The active site is a deep cleft between the N- and the C-terminal domains to which the inhibitor binds in an extended conformation filling the S4 to S2' pockets. The structure of the complex is compared with that of the related uninhibited enzyme pepsin. Significant changes in the relative orientation of the N- and C-terminal domains are observed. In the inhibited renin structure the C-terminal loop segments forming the active site are closer to those from the N-terminal domain than in the related "open" pepsin structure. In addition, the structure of uninhibited glycosylated renin has been determined at 2.8 A resolution from a cubic crystal form with two renin molecules in the asymmetric unit. The two independent renin molecules show different conformations with respect to the relative orientation of their N- and C-terminal domains; one molecule is found in the "closed inhibited" conformation, the other in the "open uninhibited" conformation.     

Induced structure of a helical switch as a mechanism to regulate enzymatic activity

Herpesviruses encode a protease that is activated by homodimerization at high enzyme concentrations during lytic replication. The homodimer contains two active sites, which are distal from the dimer interface. Assignment of backbone NMR resonances and engineering of a redox switch show that two helices position a loop containing catalytic residues within each active site.     

Structural and Kinetic Studies of the Allosteric Transition in Sulfolobus solfataricus Uracil Phosphoribosyltransferase: Permanent Activation by Engineering of the C-Terminus

Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-α-1-diphosphate (PRPP) and uracil to uridine monophosphate (UMP) and diphosphate (PP_i). The tetrameric enzyme from Sulfolobus solfataricus has a unique type of allosteric regulation by cytidine triphosphate (CTP) and guanosine triphosphate (GTP). Here we report two structures of the activated state in complex with GTP. One structure (refined at 2.8-Å resolution) contains PRPP in all active sites, while the other structure (refined at 2.9-Å resolution) has PRPP in two sites and the hydrolysis products, ribose-5-phosphate and PP_i, in the other sites. Combined with three existing structures of uracil phosphoribosyltransferase in complex with UMP and the allosteric inhibitor cytidine triphosphate (CTP), these structures provide valuable insight into the mechanism of allosteric transition from inhibited to active enzyme. The regulatory triphosphates bind at a site in the center of the tetramer in a different manner and change the quaternary arrangement. Both effectors contact Pro94 at the beginning of a long β-strand in the dimer interface, which extends into a flexible loop over the active site. In the GTP-bound state, two flexible loop residues, Tyr123 and Lys125, bind the PP_i moiety of PRPP in the neighboring subunit and contribute to catalysis, while in the inhibited state, they contribute to the configuration of the active site for UMP rather than PRPP binding. The C-terminal Gly216 participates in a hydrogen-bond network in the dimer interface that stabilizes the inhibited, but not the activated, state. Tagging the C-terminus with additional amino acids generates an endogenously activated enzyme that binds GTP without effects on activity.     

Low-resolution structure of recombinant human granulocyte-macrophage colony stimulating factor

A recombinant form of human granulocyte-macrophage colony stimulating factor (GM-CSF) which contains no carbohydrate has been crystallized. Multiple isomorphous replacement analysis using five heavy-atom derivatives has yielded an image of the structure at 6 A resolution that showed two molecules per asymmetric unit and allowed determination of the non-crystallographic symmetry transformation. The 6 A resolution result shows that the core of GM-CSF consists of four helices. The angles at which the helices pack together distinguishes this structure from known antiparallel four-helix bundle proteins. Consideration of the amino acid sequence properties and previous structural characterizations of GM-CSF leads to an assignment of the probable protein segments that form the helices.     

Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution.

The three-dimensional structure of the highly thermostable 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus has been determined by the multiple isomorphous replacement method and refined to 2.2 A resolution. The final R-factor is 0.185 for 20,307 reflections. The crystal asymmetric unit has one subunit consisting of 345 amino acid residues. The polypeptide chain of this subunit is folded into two domains (first and second domains) with parallel alpha/beta motifs. The domains are similar in their conformations and folding topologies, but differ from those of the NAD-binding domains of such well-known enzymes as the alcohol and lactate dehydrogenases. A beta-strand that is a part of the long arm-like polypeptide protruding from the second domain comes into contact with another subunit and contributes to the formation of an isologous dimer with a crystallographic 2-fold symmetry. Close subunit contacts are also present at two alpha-helices in the second domain. These helices strongly interact hydrophobically with the corresponding helices of the other subunit to form a hydrophobic core at the center of the dimer. Two large pockets that exist between the first domain of one subunit and the second domain of the other include the amino acid residues responsible for substrate binding. These results indicate that the dimeric form is essential for the IPMDH to express enzymatic activity and that the close subunit contact at the hydrophobic core is important for the thermal stability of the enzyme.     

Three-dimensional structure of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred reaction mechanism

2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 A resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase. Its closest structural homologue is E. coli 8-amino-7-oxononanoate synthase. Like many other members of the alpha family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal. There are two active sites per dimer, located at the dimer interface. Both monomers contribute side chains to each active/substrate binding site. Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure. The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL. A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.