Infrared spectroscopic study of the metal-coordination structures of calcium-binding proteins

Carboxylate (COO⁻) groups can coordinate to metal ions in of the following four modes: ‘unidentate’, ‘bidentate’, ‘bridging’ and ‘pseudo-bridging’ modes. COO⁻ stretching frequencies provide information about the coordination modes of COO⁻ groups to metal ions. We review the Fourier-transform infrared spectroscopy (FTIR) of side-chain COO⁻ groups of Ca²⁺-binding proteins: pike parvalbumin pI 4.10, bovine calmodulin and Akazara scallop troponin C. FTIR spectroscopy of Akazara scallop troponin C has demonstrated that the coordination structure of Mg²⁺ is distinctly different from that of Ca²⁺ in the Ca²⁺-binding site. The assignments of the COO⁻ antisymmetric stretch have been ensured on the basis of the spectra of calcium-binding peptide analogues. The downshift of the COO⁻ antisymmetric stretching mode from 1565 cm^-1 to 1555-1540 cm⁻¹ upon Ca²⁺ binding is a commonly observed feature of FTIR spectra for EF-hand proteins.     

Design of Ca2+‐independent Staphylococcus aureus sortase A mutants

The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca²⁺, because binding of Ca²⁺ to Glu residues distal to the active site stabilizes the substrate binding site. To obtain Ca²⁺‐independent SaSrtA, we substituted two Glu residues in the Ca²⁺‐binding pocket (Glu¹⁰⁵ and Glu¹⁰⁸). Although single mutations decreased SaSrtA activity, mutations of both Glu¹⁰⁵ and Glu¹⁰⁸ resulted in Ca²⁺‐independent activity. Kinetic analysis suggested that the double mutations affect the substrate binding site, without affecting substrate specificity. This approach will allow us to develop SaSrtA variants suitable for various applications, including in vivo site‐specific protein modification and labeling. Biotechnol. Bioeng. 2012; 109: 2955–2961. © 2012 Wiley Periodicals, Inc.     

Coordination structures of Mg2+ and Ca2+ in three types of tobacco calmodulins in solution: Fourier‐transform infrared spectroscopic studies of side‐chain COO− groups

Calmodulin (CaM) is a Ca²⁺‐binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier‐transform infrared spectroscopy to compare the secondary and coordination structures of Mg²⁺ and Ca²⁺ among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak β‐strand bands and the weak 1661 cm^?1 band. Coordination structures of Mg²⁺ of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca²⁺‐binding manner was similar among the three CaMs. The amplitude differences of the band at 1554–1550 cm^?1 obtained by second‐derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca²⁺] increases under low [Ca²⁺] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca²⁺]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg²⁺‐binding manner and needs higher [Ca²⁺] for bidentate Ca²⁺ coordination of 12th Glu in EF‐hand motifs. The Ca²⁺‐binding mechanisms of the EF‐hand motifs of the three CaMs are similar; however, the cation‐dependent conformational change in NtCaM13 is unique among the three NtCaMs. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 472–483, 2013.     

A charge‐sensitive loop in the FKBP38 catalytic domain modulates Bcl‐2 binding

The Bcl‐2 inhibitor FKBP38 is regulated by the Ca²⁺‐sensor calmodulin (CaM). Here we show a hitherto unknown low‐affinity cation‐binding site in the FKBP domain of FKBP38, which may afford an additional level of regulation based on electrostatic interactions. Fluorescence titration experiments indicate that in particular the physiologically relevant Ca²⁺ ion binds to this site. NMR‐based chemical shift perturbation data locate this cation‐interaction site within the β5–α1 loop (Leu90–Ile96) of the FKBP domain, which contains the acidic Asp92 and Asp94 side‐chains. Binding constants were subsequently determined for K⁺, Mg²⁺, Ca²⁺, and La³⁺, indicating that the net charge and the radius of the ion influences the binding interaction. X‐ray diffraction data furthermore show that the conformation of the β5–α1 loop is influenced by the presence of a positively charged guanidinium group belonging to a neighboring FKBP38 molecule in the crystal lattice. The position of the cation‐binding site has been further elucidated based on pseudocontact shift data obtained by NMR via titration with Tb³⁺. Elimination of the Ca²⁺‐binding capacity by substitution of the respective aspartate residues in a D92N/D94N double‐substituted variant reduces the Bcl‐2 affinity of the FKBP38^35–153/CaM complex to the same degree as the presence of Ca²⁺ in the wild‐type protein. Hence, this charge‐sensitive site in the FKBP domain participates in the regulation of FKBP38 function by enabling electrostatic interactions with ligand proteins and/or salt ions such as Ca²⁺. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of Two EF‐hand Domain‐containing Proteins from Toxoplasma gondii

The universal role of calcium (Ca²⁺) as a second messenger in cells depends on a large number of Ca²⁺‐binding proteins (CBP), which are able to bind Ca²⁺ through specific domains. Many CBPs share a type of Ca²⁺‐binding domain known as the EF‐hand. The EF‐hand motif has been well studied and consists of a helix‐loop‐helix structural domain with specific amino acids in the loop region that interact with Ca²⁺. In Toxoplasma gondii a large number of genes (approximately 68) are predicted to have at least one EF‐hand motif. The majority of these genes have not been characterized. We report the characterization of two EF‐hand motif‐containing proteins, TgGT1_216620 and TgGT1_280480, which localize to the plasma membrane and to the rhoptry bulb, respectively. Genetic disruption of these genes by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated protein 9) resulted in mutant parasite clones (Δtg216620 and Δtg280480) that grew at a slower rate than control cells. Ca²⁺ measurements showed that Δtg216620 cells did not respond to extracellular Ca²⁺ as the parental controls while Δtg280480 cells appeared to respond as the parental cells. Our hypothesis is that TgGT1_216620 is important for Ca²⁺ influx while TgGT1_280480 may be playing a different role in the rhoptries.     

New insight into the binding mode of peptides at urotensin‐II receptor by Trp‐constrained analogues of P5U and urantide

Urotensin II (U‐II) is a disulfide bridged peptide hormone identified as the ligand of a G‐protein‐coupled receptor. Human U‐II (H‐Glu‐Thr‐Pro‐Asp‐c[Cys‐Phe‐Trp‐Lys‐Tyr‐Cys]‐Val‐OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of human U‐II termed P5U (H‐Asp‐c[Pen‐Phe‐Trp‐Lys‐Tyr‐Cys]‐Val‐OH) and the compound termed urantide (H‐Asp‐c[Pen‐Phe‐d ‐Trp‐Orn‐Tyr‐Cys]‐Val‐OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized four analogues of P5U and urantide in which the Trp⁷ residue was replaced by the highly constrained l ‐Tpi and d ‐Tpi residues. The replacement of the Trp⁷ by Tpi led to active analogues. Solution NMR analysis allowed improving the knowledge on conformation–activity relationships previously reported on UT receptor ligands. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Fe2+ binding on amyloid β‐peptide promotes aggregation

The metal ions Zn²⁺, Cu²⁺, and Fe²⁺ play a significant role in the aggregation mechanism of Aβ peptides. However, the nature of binding between metal and peptide has remained elusive; the detailed information on this from the experimental study is very difficult. Density functional theory (dft) (M06‐2X/6‐311++G (2df,2pd) +LANL2DZ) has employed to determine the force field resulting due to metal and histidine interaction. We performed 200 ns molecular dynamics (MD) simulation on Aβ_1‐42‐Zn²⁺, Aβ_1‐42‐Cu²⁺, and Aβ_1‐42‐Fe²⁺ systems in explicit water with different combination of coordinating residues including the three Histidine residues in the N‐terminal. The present investigation, the Aβ_1‐42‐Zn²⁺ system possess three turn conformations separated by coil structure. Zn²⁺ binding caused the loss of the helical structure of N‐terminal residues which transformed into the S‐shaped conformation. Zn²⁺ has reduced the coil and increases the turn content of the peptide compared with experimental study. On the other hand, the Cu²⁺ binds with peptide, β sheet formation is observed at the N‐terminal residues of the peptide. Fe²⁺ binding is to promote the formation of Glu22‐Lys28 salt‐bridge which stabilized the turn conformation in the Phe19‐Gly25 residues, subsequently β sheets were observed at His13‐Lys18 and Gly29‐Gly37 residues. The turn conformation facilitates the β sheets are arranged in parallel by enhancing the hydrophobic contact between Gly25 and Met35, Lys16 and Met35, Leu17 and Leu34, Val18 and Leu34 residues. The Fe²⁺ binding reduced the helix structure and increases the β sheet content in the peptide, which suggested, Fe²⁺ promotes the oligomerization by enhancing the peptide‐peptide interaction. Proteins 2016; 84:1257–1274. © 2016 Wiley Periodicals, Inc.     

Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short‐term synaptic plasticity

Ca²⁺ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca²⁺–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca²⁺ signals. We solved the structure of Ca²⁺₄–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca²⁺₂–CaM complex. The Ca²⁺ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca²⁺–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca²⁺]_i, whereas the N‐module acts as a sensor at micromolar [Ca²⁺]_i. This Ca²⁺/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca²⁺‐dependent modulation of short‐term synaptic plasticity.     

Binding of calcium is sensed structurally and dynamically throughout the second calcium‐binding domain of the sodium/calcium exchanger

We report the effects of Ca²⁺ binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca²⁺‐binding domain (CBD2) of the sodium–calcium exchanger. Analysis of the Ca²⁺‐induced chemical shifts perturbations yields similar K_D values of 16–24 μM for the two CBD2‐AD Ca²⁺‐binding sites, and significant effects are observed up to 20 Å away. To quantify the Ca²⁺‐induced chemical shift changes, we performed a comparative analysis of eight Ca²⁺‐binding proteins that revealed large differences between different protein folds. The CBD2 ¹⁵N relaxation data show the CBD2‐AD Ca²⁺ coordinating loops to be more rigid in the Ca²⁺‐bound state as well as to affect the FG‐loop located at the opposite site of the domain. The equivalent loops of the CBD2‐BD splice variant do not bind Ca²⁺ and are much more dynamic relative to both the Ca²⁺‐bound and apo forms of CBD2‐AD. A more structured FG‐loop in CBD2‐BD is suggested by increased S² order parameter values relative to both forms of CBD2‐AD. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca²⁺‐binding event is felt throughout the molecule. The data suggest that the FG‐loop plays an important role in connecting the Ca²⁺‐binding event with the other cytosolic domains of the NCX, in line with in vivo and in vitro biochemical data as well as modeling results that connect the CBD2 FG‐loop with the first Ca²⁺‐binding domain of NCX. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Predicting Ca2+‐binding sites using refined carbon clusters

Identifying Ca²⁺‐binding sites in proteins is the first step toward understanding the molecular basis of diseases related to Ca²⁺‐binding proteins. Currently, these sites are identified in structures either through X‐ray crystallography or NMR analysis. However, Ca²⁺‐binding sites are not always visible in X‐ray structures due to flexibility in the binding region or low occupancy in a Ca²⁺‐binding site. Similarly, both Ca²⁺ and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca²⁺‐binding sites in both X‐ray and NMR structures, we report a new graph theory algorithm (MUG^C) to predict Ca²⁺‐binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side‐chain oxygen ligand co‐ordinates, MUG^C is able to achieve 94% sensitivity with 76% selectivity on a dataset of X‐ray structures composed of 43 Ca²⁺‐binding proteins. Additionally, prediction of Ca²⁺‐binding sites in NMR structures was obtained by MUG^C using a different set of parameters, which were determined by the analysis of both Ca²⁺‐constrained and unconstrained Ca²⁺‐loaded structures derived from NMR data. MUG^C identified 20 of 21 Ca²⁺‐binding sites in NMR structures inferred without the use of Ca²⁺ constraints. MUG^C predictions are also highly selective for Ca²⁺‐binding sites as analyses of binding sites for Mg²⁺, Zn²⁺, and Pb²⁺ were not identified as Ca²⁺‐binding sites. These results indicate that the geometric arrangement of the second‐shell carbon cluster is sufficient not only for accurate identification of Ca²⁺‐binding sites in NMR and X‐ray structures but also for selective differentiation between Ca²⁺ and other relevant divalent cations. © Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Enhanced dissolution of the substrate d,l‐2‐amino‐Δ2‐thiazoline‐4‐carboxylic acid and enzymatic production of l‐cysteine at high concentrations

l ‐Cysteine is widely used as a precursor in the pharmaceutical, cosmetic, food, and feed additive industries. It has been industrially produced from hydrolysis of human and animal hairs, which is limited for industrial production. At the same time, chemical hydrolysis causes the formation of intractable waste material. Thus, environmentally friendly methods have been developed. A big obstacle of currently available methods is the low substrate solubility leading to poor l ‐cysteine yield. Here, a method for improving the low solubility of the substrate d ,l ‐2‐amino‐Δ²‐thiazoline‐4‐carboxylic acid (d ,l ‐ATC) is presented and the enzymatic reaction at high concentration levels was optimized. The substrate was dissolved in large amounts in aqueous solutions by pH control using salts. d ,l ‐ATC solubility increased with an increasing solution pH due to its enhanced hydrophilicity, which can be achieved by a shift to dissociated carboxylic group (–COO^?). The highest d ,l ‐ATC solubility of 610 mM was obtained at pH 10.5. The maximum l ‐cysteine yield of 250 mM was attained at pH 9.1, which lies between the optimum values for high substrate solubility and reaction rate. The product yield could be increased by more than 10 times compared to those in previous reports, which is industrially meaningful.     

Evaluation of substrate promiscuity of an L‐carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43

N‐carbamoyl‐amino‐acid amidohydrolase (also known as N‐carbamoylase) is the stereospecific enzyme responsible for the chirality of the D ‐ or L ‐amino acid obtained in the “Hydantoinase Process.” This process is based on the dynamic kinetic resolution of D ,L ‐5‐monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L ‐N‐carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N‐acetyl and N‐formyl‐L ‐amino acids as well as the known N‐carbamoyl‐L ‐amino acids, thus proving its substrate promiscuity. BsLcar showed faster hydrolysis for N‐formyl‐L ‐amino acids than for N‐carbamoyl and N‐acetyl‐L ‐derivatives, with a catalytic efficiency (k_cat/K_m) of 8.58 × 10⁵, 1.83 × 10⁴, and 1.78 × 10³ (s^?1 M^?1), respectively, for the three precursors of L ‐methionine. Optimum reaction conditions for BsLcar, using the three N‐substituted‐L ‐methionine substrates, were 65°C and pH 7.5. In all three cases, the metal ions Co²⁺, Mn²⁺, and Ni²⁺ greatly enhanced BsLcar activity, whereas metal‐chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co²⁺‐dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Direct and indirect roles of His‐418 in metal binding and in the activity of β‐galactosidase (E. coli)

The active site of ß‐galactosidase (E. coli) contains a Mg²⁺ ion ligated by Glu‐416, His‐418 and Glu‐461 plus three water molecules. A Na⁺ ion binds nearby. To better understand the role of the active site Mg²⁺ and its ligands, His‐418 was substituted with Asn, Glu and Phe. The Asn‐418 and Glu‐418 variants could be crystallized and the structures were shown to be very similar to native enzyme. The Glu‐418 variant showed increased mobility of some residues in the active site, which explains why the substitutions at the Mg²⁺ site also reduce Na⁺ binding affinity. The Phe variant had reduced stability, bound Mg²⁺ weakly and could not be crystallized. All three variants have low catalytic activity due to large decreases in the degalactosylation rate. Large decreases in substrate binding affinity were also observed but transition state analogs bound as well or better than to native. The results indicate that His‐418, together with the Mg²⁺, modulate the central role of Glu‐461 in binding and as a general acid/base catalyst in the overall catalytic mechanism. Glucose binding as an acceptor was also dramatically decreased, indicating that His‐418 is very important for the formation of allolactose (the natural inducer of the lac operon).     

Synthesis and biological evaluation of new active For‐Met‐Leu‐Phe‐OMe analogues containing para‐substituted Phe residues

In the present study, we report synthesis and biological evaluation of the N‐Boc‐protected tripeptides 4a–l and N‐For protected tripeptides 5a–l as new For‐Met‐Leu‐Phe‐OMe (fMLF‐OMe) analogues. All the new ligands are characterized by the C‐terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a–l and the antagonism of 4a–l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF‐OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with ‐CH₃ and ‐C(CH₃)₃, respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i , containing ‐F and ‐I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Epitope analysis of the multiphosphorylated peptide αs1‐casein(59–79)

The multiphosphorylated tryptic peptide α_s1‐casein(59–79) has been shown to be antigenic with anti‐casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With α_s1‐casein(59–79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding of the anti‐casein antibodies. Peptides Glu‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu and Ile‐Val‐Pro‐Asn‐Ser(P)‐Val‐Glu‐Glu inhibited antibody binding by 20.0±3.6% and 60.3±7.9%, respectively. The epitope of Glu⁶³‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu⁷⁰ was further localised to the phosphoseryl cluster as the peptide Ser(P)‐Ser(P)‐Ser(P) significantly inhibited binding of the anti‐casein antibodies to α_s1‐casein(59–79) by 29.5±7.4%. Substitution of Ser(P)⁷⁵ with Ser⁷⁵ in the second inhibitory peptide Ile‐Val‐Pro‐Asn‐Ser(P)⁷⁵‐Val‐Glu‐Glu also abolished inhibition of antibody binding to α_s1‐casein (59–79) demonstrating that Ser(P)⁷⁵ is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence ‐Ser(P)⁶⁶‐Ser(P)‐Ser(P)⁶⁸ and in the sequence ‐Pro⁷³‐Asn‐Ser(P)‐Val‐Glu⁷⁷‐ are critical for antibody binding to α_s1‐casein(59–79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Structure‐based investigation into the functional roles of the extended loop and substrate‐recognition sites in an endo‐β‐1,4‐d‐mannanase from the Antarctic springtail,Cryptopygus antarcticus

Endo‐β‐1,4‐d ‐mannanase from the Antarctic springtail, Cryptopygus antarcticus (CaMan), is a cold‐adapted β‐mannanase that has the lowest optimum temperature (30°C) of all known β‐mannanases. Here, we report the apo‐ and mannopentaose (M5) complex structures of CaMan. Structural comparison of CaMan with other β‐mannanases from the multicellular animals reveals that CaMan has an extended loop that alters topography of the active site. Structural and mutational analyses suggest that this extended loop is linked to the cold‐adapted enzymatic activity. From the CaMan‐M5 complex structure, we defined the mannose‐recognition subsites and observed unreported M5 binding site on the surface of CaMan. Proteins 2014; 82:3217–3223. © 2014 Wiley Periodicals, Inc.     

Conformational transformation of ascidiacyclamide analogues induced by incorporating enantiomers of phenylalanine, 1‐naphthylalanine or 2‐naphthylalanine

We designed five ascidiacyclamide analogues [cyclo(‐Xxx¹‐oxazoline²‐d ‐Val³‐thiazole⁴‐l ‐Ile⁵‐oxazoline⁶‐d ‐Val⁷‐thiazole⁸‐)] incorporating l ‐1‐naphthylalanine (l ‐1Nal), l ‐2‐naphthylalanine (l ‐2Nal), d ‐phenylalanine (d ‐Phe), d ‐1‐naphthylalanine (d ‐1Nal) or d ‐2‐naphthylalanine (d ‐2Nal) into the Xxx¹ position of the peptide. The conformations of these analogues were then examined using ¹H NMR, CD spectroscopy, and X‐ray diffraction. These analyses suggested that d ‐enantiomer‐incorporated ASCs [(d ‐Phe), (d ‐1Nal), and (d ‐2Nal)ASC] transformed from the folded to the open structure in solution more easily than l ‐enantiomer‐incorporated ASCs [(l ‐Phe), (l ‐1Nal), and (l ‐2Nal)ASC]. Structural comparison of the two analogues containing isomeric naphthyl groups showed that the 1‐naphthyl isomer induced a more stable open structure than the 2‐naphthyl isomer. In particular, [d ‐1Nal]ASC showed the most significant transformation from the folded to the open structure in solution, and exhibited the strongest cytotoxicity toward HL‐60 cells. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Potential anti‐bacterial drug target: Structural characterization of 3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase from Salmonella typhimurium LT2

3,4‐Dihydroxy‐2‐butanone‐4‐phosphate synthase (DHBPS) encoded by ribB gene is one of the first enzymes in riboflavin biosynthesis pathway and catalyzes the conversion of ribulose‐5‐phosphate (Ru5P) to 3,4‐dihydroxy‐2‐butanone‐4‐phosphate and formate. DHBPS is an attractive target for developing anti‐bacterial drugs as this enzyme is essential for pathogens, but absent in humans. The recombinant DHBPS enzyme of Salmonella requires magnesium ion for its activity and catalyzes the formation of 3,4‐dihydroxy‐2‐butanone‐4‐phosphate from Ru5P at a rate of 199 nmol min^?1 mg^?1 with K_m value of 116 μM at 37°C. Further, we have determined the crystal structures of Salmonella DHBPS in complex with sulfate, Ru5P and sulfate‐zinc ion at a resolution of 2.80, 2.52, and 1.86 Å, respectively. Analysis of these crystal structures reveals that the acidic loop (residues 34–39) responsible for the acid‐base catalysis is disordered in the absence of substrate or metal ion at the active site. Upon binding either substrate or sulfate and metal ions, the acidic loop becomes stabilized, adopts a closed conformation and interacts with the substrate. Our structure for the first time reveals that binding of substrate Ru5P alone is sufficient for the stabilization of the acidic active site loop into a closed conformation. In addition, the Glu38 residue from the acidic active site loop undergoes a conformational change upon Ru5P binding, which helps in positioning the second metal ion that stabilizes the Ru5P and the reaction intermediates. This is the first structural report of DHBPS in complex with either substrate or metal ion from any eubacteria. Proteins 2010. © 2010 Wiley‐Liss, Inc.