Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization.

[A new approach for study of structural and functional properties of proteins with unknown functions].

Selected proteins were produced in Escherichia coli bacterial expression system--three proteins from extremophil bacteria: a putative monooxygenase from Deinococcus radiodurans, a putative nucleotidyltransferase from Thermotoga maritima, a putative oxidoreductase from Exiguobacterium sibiricum; and a shaperon from Homo sapiens DJ-1. The protocol of isolation & purification of recombinant proteins were developed that allowed to obtain expression products with the purity of no less than 96%. Conditions for the crystallization have been selected that allowed a stable growth of crystals. Preliminary x-ray experiments were conducted in order to confirm the quality of produced crystals; the resolution of obtained structural data was from 1.2 to 1.8 angstrom.

To automate or not to automate: this is the question.

New protocols and instrumentation significantly boost the outcome of structural biology, which has resulted in significant growth in the number of deposited Protein Data Bank structures. However, even an enormous increase of the productivity of a single step of the structure determination process may not significantly shorten the time between clone and deposition or publication. For example, in a medium size laboratory equipped with the LabDB and HKL-3000 systems, we show that automation of some (and integration of all) steps of the X-ray structure determination pathway is critical for laboratory productivity. Moreover, we show that the lag period after which the impact of a technology change is observed is longer than expected.

Enzymatic oxidation of cadmium and lead metals photodeposited on cadmium sulfide.

Cadmium and lead metals deposited on CdS particles are shown to act as substrates--electron donors for enzymes, hydrogenase from Thiocapsa roseopersicina (HG), NAD-dependent hydrogenase from Alcaligenes eutrophus (NLH), and ferredoxin:NADP oxidoreductase (FNR) from Chlorella in the formation of hydrogen, NADH and NADPH, respectively. Adsorption of the enzyme on the surface of the metallized CdS particle is required for enzymatic oxidation of metal. The maximum rates for the formation of hydrogen and NADH catalyzed by hydrogenase and NAD-dependent hydrogenase with metals as electron donors are comparable with the rates obtained for these enzymes using soluble substrates. Kinetic analysis of the enzymatic oxidation of cadmium metal has revealed that the rate decreases mainly due to the formation of a solid product, which is supposed to be Cd(OH)2. The deceleration of lead oxidation catalyzed by hydrogenase proceeds at the expense of the inhibitory effect of the formed Pb2+. The enzymatic oxidation of electrochemically prepared cadmium metal is also shown. Based on these results, a new mechanism of action of the enzymes involved in anaerobic biocorrosion is proposed. By this mechanism, the enzyme accelerates the process of metal dissolution through a mediatorless catalysis of the reduction of the enzyme substrate.

Structure of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: comparison of the Mn(2+)*2-phosphoglycolate and the Pb(2+)*2-phosphoenolpyruvate complexes and implications for catalysis.

The crystal structure of the phenylalanine-regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) from Escherichia coli in complex with Mn(2+) and the substrate analog, 2-phosphoglycolate (PGL), was determined by molecular replacement using X-ray diffraction data to 2.0 A resolution. DAHPS*Mn*PGL crystallizes in space group C2 (a=210.4 A, b=53.2 A, c=149.4 A, beta=116.1 degrees ) with its four (beta/alpha)(8) barrel subunits related by non-crystallographic 222 symmetry. The refinement was carried out without non-crystallographic symmetry restraints and yielded agreement factors of R=20.9 % and R(free)=23.9 %. Mn(2+), the most efficient metal activator, is coordinated by the same four side-chains (Cys61, His268, Glu302 and Asp326) as is the poorly activating Pb(2+). A fifth ligand is a well-defined water molecule, which is within hydrogen bonding distance to an essential lysine residue (Lys97). The distorted octahedral coordination sphere of the metal is completed by PGL, which replaces the substrate, 2-phosphoenolpyruvate (PEP), in the active site. However, unlike PEP in the Pb*PEP complex, PGL binds the Mn(2+) via one of its carboxylate oxygen atoms. A model of the active site is discussed in which PEP binds in the same orientation as does PGL in the DAHPS*Mn*PGL structure and the phosphate of E4P is tethered at the site of a bound sulfate anion. The re face of E4P can be positioned to interact with the si face of PEP with only small movement of the protein.

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.

Purification, crystallization, and preliminary crystallographic analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The phenylalanine-regulated isozyme of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) from Escherichia coli, its binary complexes with either substrate, phosphoenolpyruvate (PEP), or feedback inhibitor, Phe, and its ternary complexes with either PEP or Phe plus metal cofactor (either Mn2+, Cd2+, or Pb2+) were crystallized from polyethylglycol (PEG) solutions. All crystals of the DAHPS without Phe belong to space group C2, with cell parameters a = 213.5 A, b = 54.3 A, c = 149.0 A, beta = 116.6 degrees. All crystals of the enzyme with Phe also belong to space group C2, but with cell parameters a = 297.1 A, b = 91.4 A, c = 256.5 A, and beta = 148.2 degrees.

Metal as a novel type of the enzyme substrate. Metallic cadmium photogenerated in the system CdS-formate as a substrate of the NAD-dependent hydrogenase.

The process of NAD+ photoreduction under the coupled action of CdS semiconductor and NAD-dependent hydrogenase from hydrogen-oxidizing bacterium Alcaligenes eutrophus may be divided into light and dark stages. At the first stage, illumination of the system leads to the photooxidation of the sacrificial electron donor and results in the reduction of the semiconductor surface. At the second dark stage NAD+ is reduced to NADH in the presence of hydrogenase. Atoms of metallic Cd(0) are shown to be the true substrate of the enzymatic reaction. The prerequisite for the electron transfer from Cd(0) to hydrogenase is enzyme adsorption on the semiconductor surface. The redox center of the hydrogenase reacting with Cd(0) atoms resides on the flavin-containing heterodimer of the protein. The activity of the hydrogenase immobilized on CdS in the reaction of NAD+ reduction by metallic Cd is close to the enzyme activity with the physiological substrates in solution. Thus, the first example of a metal being the substrate of the enzymatic process is presented.

Photogeneration of NADH under coupled action of CdS semiconductor and hydrogenase from Alcaligenes eutrophus without exogenous mediators.

Photoreduction of NAD has been accomplished by a system consisting of the NAD-dependent hydrogenase from Alcaligenes eutrophus immobilized on CdS particles with formate as artificial electron donor. Enzymatically active NADH is formed under illumination of this system by visible light. Accumulation of the coenzyme dimer (NAD)2 was not detected. NAD photoreduction is supposed to proceed via the direct electron transfer from the semiconductor to the enzyme electron transport chain. However, NADH formation as a result of hydrogenase interaction with anion-radicals (CO2.-) formed in the course of formate photooxidation cannot at present be excluded.

[NAD-dependent formate dehydrogenase from methylotrophic bacteria Pseudomonas sp. 101. I. Amino acid sequence]. / NAD-zavisimaia formiatdegidrogenaza metilotrofnykh bakterii Pseudomonas sp. 101. I. Aminokislotnaia posledovatel'nost'.

The primary structure of NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 is determined. The enzyme is composed of two identical subunits, each comprising 393 amino acid residues, and has a molecular weight of 43.1 kD. To elucidate the protein's amino acid sequence, four types of digestion were used: cyanogen bromide cleavage at methionine residues, endoproteinase Lys-C digestion at lysine residues, endoproteinase Glu-C cleavage at glutamic acid residues, and tryptic digestion. The peptides obtained were purified to homogeneity and characterized.

[NAD-dependent formate dehydrogenase of methylotrophic bacteria Pseudomonas sp. 101. III. Comparative analysis]. / NAD-zavisimaia formiatdegidrogenaza metilotrofnykh bakterii Pseudomonas sp. 101. III. Sravnitel'nyi analiz.

The comparative analysis of the primary and tertiary structures of NAD-dependent bacterial formate dehydrogenase (FDH) from methylotrophic bacterium Pseudomonas sp. 101 and a number of structurally characterized NAD-dependent dehydrogenases were performed. FDH has a highly conservative fold of the coenzyme binding domain. Position of the symmetry axis in the FDH molecule relative to the beta-sheets of its coenzyme binding domain with the respective sequences of the other NAD-dependent enzymes was performed on the basis of the spatial homology between these structures. Only one of the three amino acid residues previously thought to be conserved in the coenzyme binding domains of NAD-dependent dehydrogenases is preserved in the FDH molecule (Asp-221). Two glycine residues found in all previously studied dehydrogenases are substituted in FDH by Ala-198 and Pro-256, respectively. Position of the essential thiol of FDH (Cys-255) in the protein structure was established. It is suggested that Cys-255 is situated on or near polypeptide locus taking part in the conformational changes of the protein in the course of the catalysis.

Mapping of the immunodominant regions of the NAD-dependent formate dehydrogenase.

A panel of 4 monoclonal antibodies and 7 polyclonal antisera against NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 has been obtained. The reactivity of the 37 overlapping proteolytic peptides with the monoclonal antibodies and polyclonal antisera has been studied with ELISA test. The data obtained were interpreted residing on the structural model of the formate dehydrogenase at 3 A resolution. The immunodominant regions in the formate dehydrogenase molecule and the epitopes for the monoclonal antibodies were elucidated.