Interaction of human 3-phosphoglycerate kinase with L-ADP, the mirror image of D-ADP

l-Nucleoside-analogues, mirror images of the natural d-nucleosides, are a new class of antiviral and anticancer agents. In the cell they have to be phosphorylated to pharmacologically active triphosphate forms, the last step seems to involve human 3-phosphoglycerate kinase (hPGK). Here we present a steady state kinetic and biophysical study of the interaction of the model compound l-MgADP with hPGK. l-MgADP is a good substrate with k_cat and K_m values of 685 s⁻¹ and 0.27 mM, respectively. Double inhibition studies suggest that l-MgADP binds to the specific adenosine-binding site and protects the conformation of hPGK molecule against heat denaturation, as detected by microcalorimetry. Structural details of the interaction in the enzyme active site are different for the d- and l-enantiomers (e.g. the effect of Mg²⁺), but these differences do not prevent the occurrence of the catalytic cycle, which is accompanied by the hinge-bending domain closure, as indicated by SAXS measurements.     

Role of side-chains in the operation of the main molecular hinge of 3-phosphoglycerate kinase

The single mutants (F165A, E192A, F196A, S392A, T393A) at and near the main hinge (beta-strand L) of human 3-phosphoglycerate kinase (hPGK) exhibit variously reduced enzyme activity, indicating the cumulative effects of these residues in regulating domain movements. The residues F165 and E192 are also essential in maintaining the conformational integrity of the whole molecule, including the hinge-region. Shortening of betaL by deleting T393 has led to a dramatic activity loss and the concomitant absence of domain closure (as detected by small angle X-ray scattering), demonstrating the role of betaL in functioning of hPGK. The role of each residue in the conformational transmission is described.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes

D-psicose, a rare sugar produced by the enzymatic reaction of D-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of D-fructose and D-psicose by epimerizing the C-3 position, with marked efficiency for D-psicose. The crystal structures of DPEase and its complex with the true substrate D-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of D-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the beta4-alpha4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound D-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.     

Domain closure,substrate specificity and catalysis of D-lactate dehydrogenase from Lactobacillus bulgaricus

NAD-dependent Lactobacillus bulgaricus D-Lactate dehydrogenase (D-LDHb) catalyses the reversible conversion of pyruvate into D-lactate. Crystals of D-LDHb complexed with NADH were grown and X-ray data collected to 2.2 A. The structure of D-LDHb was solved by molecular replacement using the dimeric Lactobacillus helveticus D-LDH as a model and was refined to an R-factor of 20.7%. The two subunits of the enzyme display strong asymmetry due to different crystal environments. The opening angles of the two catalytic domains with respect to the core coenzyme binding domains differ by 16 degrees. Subunit A is in an "open" conformation typical for a dehydrogenase apo enzyme and subunit B is "closed". The NADH-binding site in subunit A is only 30% occupied, while in subunit B it is fully occupied and there is a sulphate ion in the substrate-binding pocket. A pyruvate molecule has been modelled in the active site and its orientation is in agreement with existing kinetic and structural data. On domain closure, a cluster of hydrophobic residues packs tightly around the methyl group of the modelled pyruvate molecule. At least three residues from this cluster govern the substrate specificity. Substrate binding itself contributes to the stabilisation of domain closure and activation of the enzyme. In pyruvate reduction, D-LDH can adapt another protonated residue, a lysine residue, to accomplish the role of the acid catalyst His296. Required lowering of the lysine pK(a) value is explained on the basis of the H296K mutant structure.     

Promiscuity in the part-phosphorylative Entner-Doudoroff pathway of the archaeon Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a 'promiscuous' non-phosphorylative variant of the Entner-Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of D-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose.     

Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidase

During the final stages of cell-wall synthesis in bacteria, penicillin-binding proteins (PBPs) catalyse the cross-linking of peptide chains from adjacent glycan strands of nascent peptidoglycan. We have recently shown that this step can be bypassed by an L,D-transpeptidase, which confers high-level beta-lactam-resistance in Enterococcus faecium. The resistance bypass leads to replacement of D-Ala4-->D-Asx-L-Lys3 cross-links generated by the PBPs by L-Lys3-->D-Asx-L-Lys3 cross-links generated by the L,D-transpeptidase. As the first structure of a member of this new transpeptidase family, we have determined the crystal structure of a fragment of the L,D-transpeptidase from E.faecium (Ldt(fm217)) at 2.4A resolution. Ldt(fm217) consists of two domains, the N-terminal domain, a new mixed alpha-beta fold, and the ErfK_YbiS_YhnG C-terminal domain, a representative of the mainly beta class of protein structures. Residue Cys442 of the C-terminal domain has been proposed to be the catalytic residue implicated in the cleavage of the L-Lys-D-Ala peptide bond. Surface analysis of Ldt(fm217) reveals that residue Cys442 is localized in a buried pocket and is accessible by two paths on different sides of the protein. We propose that the two paths to the catalytic residue Cys442 are the binding sites for the acceptor and donor substrates of the L,D-transpeptidase.     

The transmembrane domain provides nucleotide binding specificity to the bacterial conjugation protein TrwB

In order to understand the functional significance of the transmembrane domain of TrwB, an integral membrane protein involved in bacterial conjugation, the protein was purified in the native, and also as a truncated soluble form (TrwBΔN70). The intact protein (TrwB) binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo- over deoxyribonucleotides. In contrast, TrwBΔN70 binds uniformly all tested nucleotides. The transmembrane domain has the general effect of making the nucleotide binding site(s) less accessible, but more selective. This is in contrast to other membrane proteins in which most of the protein mass, including the catalytic domain, is outside the membrane, but whose activity is not modified by the presence or absence of the transmembrane segment.     

Crystal Structure Analysis of Free and Substrate-Bound 6-Hydroxy-l-Nicotine Oxidase from Arthrobacter nicotinovorans

The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-l-nicotine oxidase (6HLNO) and 6-hydroxy-d-nicotine oxidase, which act with absolute stereospecificity on the l- and d-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 Å resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit-subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-l-nicotine at 2.05 Å resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-d-nicotine oxidase, based on models of complexes with the d-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.     

D-ribose-5-phosphate isomerase B from Escherichia coli is also a functional D-allose-6-phosphate isomerase, while the Mycobacterium tuberculosis enzyme is not

Interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate is an important step in the pentose phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars d-allose-6-phosphate (All6P) and d-allulose-6-phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme's primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-d-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme's structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate ribose-5-phosphate, shed additional light on the reaction mechanism of RpiBs generally.     

Recognition of selected monosaccharides by Pseudomonas aeruginosa Lectin II analyzed by molecular dynamics and free energy calculations

In this study, interactions of selected monosaccharides with the Pseudomonas aeruginosa Lectin II (PA-IIL) are analyzed in detail. An interesting feature of the PA-IIL binding is that the monosaccharide is interacting via two calcium ions and the binding is unusually strong for protein-saccharide interaction. We have used Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) and normal mode analysis to calculate the free energy of binding. The impact of intramolecular hydrogen bond network for the lectin/monosaccharide interaction is also analyzed.     

The flexible attachment of the N-domains to the ClpA ring body allows their use on demand

ClpA is an Hsp100 chaperone that uses the chemical energy of ATP to remodel various protein substrates to prepare them for degradation. It comprises two AAA+ modules and the N-domain, which is attached N-terminally to the first AAA+ module through a linker. On the basis of cryo-electron microscopic and X-ray crystallographic data it has been suggested that the linker confers mobility to the N-domain. In order to define the role of the N-domain in ClpAP-dependent substrate degradation we have generated a ΔN variant at the protein level by introducing a protease cleavage site. The ClpA molecule generated in this way lacks the N-domain and the associated linker but is impaired only slightly in the processing of substrates that are degraded independently of ClpS. In fact, it shows increased catalytic efficiency in the degradation of ssrA-tagged GFP compared to ClpAwt. The role of the linker attaching the N-domain to the bulk of the molecule was probed by characterizing variants with different lengths of the linker. The degradation efficiency of a ClpS-dependent N-end rule substrate, FRliGFP, is reduced for linkers that are shorter or longer than natural linkers but remains the same for the variant where the linker is replaced by an engineered sequence of equivalent length. These results suggest that the flexible attachment of the N-domains to ClpA allows their recruitment to the pore on demand for certain substrates, while allowing them to move out of the way for substrates binding directly to the pore.     

The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc1 complexes

A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a_s-type with reduction potentials of + 200, + 400 and + 160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a_s, and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be + 320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane α-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc₁ complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc₁ complexes of bacteria and mitochondria, however with distinct subunits and heme types.     

Thermodynamic analysis of mutant lac repressors

The lactose (lac) repressor is an allosteric protein that can respond to environmental changes. Mutations introduced into the DNA binding domain and the effector binding pocket affect the repressor's ability to respond to its environment. We have demonstrated how the observed phenotype is a consequence of altering the thermodynamic equilibrium constants. We discuss mutant repressors, which (1) show tighter repression; (2) induce with a previously noninducing species, orthonitrophenyl-β-d-galactoside; and (3) transform an inducible switch to one that is corepressed. The ability of point mutations to change multiple thermodynamic constants, and hence drastically alter the repressor's phenotype, shows how allosteric proteins can perform a wide array of similar yet distinct functions such as that exhibited in the Lac/Gal family of bacterial repressors.     

Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase

Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 A resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme+NADPH+reduced substrate) complex, and the other a binary (enzyme+NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2.     

Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding

l-Alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to l-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 Å resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16° toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.     

Tridec-1-ene-3,5,7,9,11-pentayne,an Ovicidal Substance from Xanthium canadense

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC. 1.4.3.5) has been purified from dry baker’s yeast to an apparent homogeneity on a polyacrylamide disc gel electrophoresis in the presence of 10 µm of phenylmethylsulfonyl fluoride throughout purification.

1) The purified enzyme, obtained as holo-flavoprotein, has a specific activity of 27µmol/mg/hr for pyridoxamine 5′-phosphate at 37°C, and a ratio of pyridoxine 5′-phosphate oxidase to pyridoxamine 5′-phosphate oxidase is approximately 0.25 at a substrate concentration of 285 µm. Km values for both substrates are 18 µm for pyridoxamine 5′-phosphate and 2.7 µm for pyridoxine 5′-phosphate, respectively.

2) The enzyme can easily oxidize pyridoxamine 5′-phosphate, but when pyridoxamine and pyridoxine 5′-phosphate are coexisted in a reaction mixture the enzyme activity is markedly suppressed much beyond the values expected from its high affinity (low Km) and low V_max for the latter substrate.

3) Optimum temperature for both substrates is approximately 45°C, and optimum pH is near 9 for pyridoxamine 5′-phosphate and 8 for pyridoxine 5′-phosphate.

4) From the data obtained, the mechanism of regulation of this enzyme in production of pyridoxal 5′-phosphate and a reasonable substrate for the enzyme in vivo are discussed.     

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.     

lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA

Most commonly used expression systems in bacteria are based on the Escherichia coli lac promoter. Furthermore, lac operon elements are used today in systems and synthetic biology. In the majority of the cases the gratuitous inducers IPTG or TMG are used. Here we report a systematic comparison of lac promoter induction by TMG and IPTG which focuses on the aspects inducer uptake, population heterogeneity and a potential influence of the transacetylase, LacA. We provide induction curves in E. coli LJ110 and in isogenic lacY and lacA mutant strains and we show that both inducers are substrates of the lactose permease at low inducer concentrations but can also enter cells independently of lactose permease if present at higher concentrations. Using a gfp reporter strain we compared TMG and IPTG induction at single cell level and showed that bimodal induction with IPTG occurred at approximately ten-fold lower concentrations than with TMG. Furthermore, we observed that lac operon induction is influenced by the transacetylase, LacA. By comparing two Plac-gfp reporter strains with and without a lacA deletion we could show that in the lacA⁺ strain the fluorescence level decreased after few hours while the fluorescence further increased in the lacA⁻ strain. The results indicate that through the activity of LacA the IPTG concentration can be reduced below an inducing threshold concentration—an influence that should be considered if low inducer amounts are used.     

A highly specific glyoxylate reductase derived from a formate dehydrogenase

A Glu141Asn mutant Paracoccus sp. 12-A formate dehydrogenase catalyzes marked glyoxylate reduction. Additional replacement of the His332-Gln313 pair with His-Glu, which is a consensus acid/base catalyst in D-hydroxyacid dehydrogenases, further improved the catalytic activity of the enzyme as to glyoxylate reduction through enhancement of the hydrogen transfer step in the catalytic process, slightly shifting the optimal pH for the reaction. On the other hand, the replacement induced no marked activity toward other 2-ketoacid substrates, and diminished the enzyme activity as to formate oxidation. Consequently, the formate dehydrogenase was converted to a highly specific and active glyoxylate reductase through only the two amino acid replacements.