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M J Jedrzejas

Publications and source records attributed to M J Jedrzejas.

At least 19 recordsLinked to original sources
PubMed2001

Conserved core structure and active site residues in alkaline phosphatase superfamily enzymes.

Cofactor-independent phosphoglycerate mutase (iPGM) has been previously identified as a member of the alkaline phosphatase (AlkP) superfamily of enzymes, based on the conservation of the predicted metal-binding residues. Structural alignment of iPGM with AlkP and cerebroside sulfatase confirmed that all these enzymes have a common core structure and revealed similarly located conserved Ser (in iPGM and AlkP) or Cys (in sulfatases) residues in their active sites. In AlkP, this Ser residue is phosphorylated during catalysis, whereas in sulfatases the active site Cys residues are modified to formylglycine and sulfatated. Similarly located Thr residue forms a phosphoenzyme intermediate in one more enzyme of the AlkP superfamily, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1 (autotaxin). Using structure-based sequence alignment, we identified homologous Ser, Thr, or Cys residues in other enzymes of the AlkP superfamily, such as phosphopentomutase, phosphoglycerol transferase, phosphonoacetate hydrolase, and GPI-anchoring enzymes (glycosylphosphatidylinositol phosphoethanolamine transferases) MCD4, GPI7, and GPI13. We predict that catalytical cycles of all the enzymes of AlkP superfamily include phosphoenzyme (or sulfoenzyme) intermediates.

Alkaline Phosphatase↗
PubMed2001

Hyaluronan binding and degradation by Streptococcus agalactiae hyaluronate lyase.

Streptococcus agalactiae hyaluronate lyase is a virulence factor that helps this pathogen to break through the biophysical barrier of the host tissues by the enzymatic degradation of hyaluronan and certain chondroitin sulfates at beta-1,4 glycosidic linkages. Crystal structures of the native enzyme and the enzyme-product complex were determined at 2.1- and 2.2-A resolutions, respectively. An elongated cleft transversing the middle of the molecule has been identified as the substrate-binding place. Two product molecules of hyaluronan degradation were observed bound to the cleft. The enzyme catalytic site was identified to comprise three residues: His(479), Tyr(488), and Asn(429). The highly positively charged cleft facilitates the binding of the negatively charged polymeric substrate chain. The matching between the aromatic patch of the enzyme and the hydrophobic patch of the substrate chain anchors the substrate chain into degradation position. A pair of proton exchanges between the enzyme and the substrate results in the cleavage of the beta-1,4 glycosidic linkage of the substrate chain and the unsaturation of the product. Phe(423) likely determines the size of the product at the product release side of the catalytic region. Hyaluronan chain is processively degraded from the reducing end toward the nonreducing end. The unsulfated or 6-sulfated regions of chondroitin sulfate can also be degraded in the same manner as hyaluronan.

Amino Acid Sequence↗
PubMed2001

Characterization of selected strains of pneumococcal surface protein A.

Several proteins, in addition to the polysaccharide capsule, have recently been implicated in the full virulence of the Streptococcus pneumoniae bacterial pathogen. One of these novel virulence factors of S. pneumoniae is pneumococcal surface protein A (PspA). The N-terminal, cell surface exposed, and functional part of PspA is essential for full pneumococcal virulence, as evidenced by the fact that antibodies raised against this part of the protein are protective against pneumococcal infections. PspA has recently been implicated in anti-complementary function as it reduces complement-mediated clearance and phagocytosis of pneumococci. Several recombinant N-terminal fragments of PspA from different strains of pneumococci, Rx1, BG9739, BG6380, EF3296, and EF5668, were analyzed using circular dichroism, analytical ultracentrifugation sedimentation velocity and equilibrium methods, and sequence homology. Uniformly, all strains of PspA molecules studied have a high alpha-helical secondary structure content and they adopt predominantly a coiled-coil structure with an elongated, likely rod-like shape. No beta-sheet structures were detected for any of the PspA molecules analyzed. All PspAs were found to be monomeric in solution with the exception of the BG9739 strain which had the propensity to partially aggregate but only into a tetrameric form. These structural properties were correlated with the functional, anti-complementary properties of PspA molecules based on the polar distribution of highly charged termini of its coiled-coil domain. The recombinant Rx1 PspA is currently under consideration for pneumococcal vaccine development.

Amino Acid Sequence↗
PubMed2001

Vitamin C inhibits the enzymatic activity of Streptococcus pneumoniae hyaluronate lyase.

Enzyme activity measurement showed that L-ascorbic acid (vitamin C (Vc)) competitively inhibits the hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. The complex crystal structure of this enzyme with Vc was determined at 2.0 A resolution. One Vc molecule was found to bind to the active site of the enzyme. The Vc carboxyl group provides the negative charges that lead the molecule into the highly positively charged cleft of the enzyme. The Vc ring system forms hydrophobic interactions with the side chain of Trp-292, which is one of the aromatic patch residues of this enzyme responsible for the selection of the cleavage sites on the substrate chain. The binding of Vc inhibits the substrate binding at hyaluronan 1, 2, and 3 (HA1, HA2, and HA3) catalytic positions. The high concentration of Vc in human tissues probably provides a low level of natural resistance to the pneumococcal invasion. This is the first time that Vc the direct inhibition on the bacterial "spreading factor" was reported, and Vc is also the first chemical that has been shown experimentally to have an inhibitory effect on bacterial hyaluronate lyase. These studies also highlight the possible structural requirement for the design of a stronger inhibitor of bacterial hyaluronate lyase.

Ascorbic Acid↗
PubMed2001

Kinetic properties of Streptococcus pneumoniae hyaluronate lyase.

Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this bacterial pathogen, which causes significant mortality and morbidity in human populations worldwide. The primary function of this enzyme is the degradation of hyaluronan, a major component of the extracellular matrix of the tissues of practically all vertebrates. The enzyme uses a processive mode of action to degrade hyaluronan to a final product, an unsaturated disaccharide hyaluronan unit. This catalysis proceeds via a five-step proton acceptance and donation mechanism that includes substrate binding, catalysis, release of the disaccharide product, translocation of the remaining hyaluronan substrate, and proton exchange with microenvironment. Based on the analysis of the three-dimensional structure of the native enzyme and its complexes with hexasaccharide substrate and disaccharide product, several residues have been chosen for mutation studies. These mutated residues included the catalytic residues Asn349, His399, Tyr408, and residues responsible for substrate binding and translocation, Arg243 and Asn580. The comparison of the kinetic properties of the wild-type with the mutant enzymes allowed for the characterization of every mutant and the correlation of the kinetic properties of the enzyme with its structure. The comparison of the wild-type hyaluronate lyase with other polysaccharide-degrading enzymes, the hydrolases endonuclease and glucoamylase, shows striking similarity of K(m)s for all of these different enzymes.

Binding Sites↗
PubMed2001

A cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus is actually a broad specificity phosphatase.

The distribution of phosphoglycerate mutase (PGM) activity in bacteria is complex, with some organisms possessing both a cofactor-dependent and a cofactor-independent PGM and others having only one of these enzymes. Although Bacillus species contain only a cofactor-independent PGM, genes homologous to those encoding cofactor-dependent PGMs have been detected in this group of bacteria, but in at least one case the encoded protein lacks significant PGM activity. Here we apply sequence analysis, molecular modeling, and enzymatic assays to the cofactor-dependent PGM homologs from B. stearothermophilus and B. subtilis, and show that these enzymes are phosphatases with broad substrate specificity. Homologs from other gram-positive bacteria are also likely to possess phosphatase activity. These studies clearly show that the exploration of genomic sequences through three-dimensional modeling is capable of producing useful predictions regarding function. However, significant methodological improvements will be needed before such analysis can be carried out automatically.

Amino Acid Sequence↗
PubMed2001

Pneumococcal virulence factors: structure and function.

The overall goal for this review is to summarize the current body of knowledge about the structure and function of major known antigens of Streptococcus pneumoniae, a major gram-positive bacterial pathogen of humans. This information is then related to the role of these proteins in pneumococcal pathogenesis and in the development of new vaccines and/or other antimicrobial agents. S. pneumoniae is the most common cause of fatal community-acquired pneumonia in the elderly and is also one of the most common causes of middle ear infections and meningitis in children. The present vaccine for the pneumococcus consists of a mixture of 23 different capsular polysaccharides. While this vaccine is very effective in young adults, who are normally at low risk of serious disease, it is only about 60% effective in the elderly. In children younger than 2 years the vaccine is ineffective and is not recommended due to the inability of this age group to mount an antibody response to the pneumococcal polysaccharides. Antimicrobial drugs such as penicillin have diminished the risk from pneumococcal disease. Several pneumococcal proteins including pneumococcal surface proteins A and C, hyaluronate lyase, pneumolysin, autolysin, pneumococcal surface antigen A, choline binding protein A, and two neuraminidase enzymes are being investigated as potential vaccine or drug targets. Essentially all of these antigens have been or are being investigated on a structural level in addition to being characterized biochemically. Recently, three-dimensional structures for hyaluronate lyase and pneumococcal surface antigen A became available from X-ray crystallography determinations. Also, modeling studies based on biophysical measurements provided more information about the structures of pneumolysin and pneumococcal surface protein A. Structural and biochemical studies of these pneumococcal virulence factors have facilitated the development of novel antibiotics or protein antigen-based vaccines as an alternative to polysaccharide-based vaccines for the treatment of pneumococcal disease.

Amino Acid Sequence↗
PubMed2000

Mechanism of catalysis of the cofactor-independent phosphoglycerate mutase from Bacillus stearothermophilus. Crystal structure of the complex with 2-phosphoglycerate.

The structure of the complex between the 2, 3-diphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus and its 3-phosphoglycerate substrate has recently been solved, and analysis of this structure allowed formulation of a mechanism for iPGM catalysis. In order to obtain further evidence for this mechanism, we have solved the structure of this iPGM complexed with 2-phosphoglycerate and two Mn(2+) ions at 1. 7-A resolution. The structure consists of two different domains connected by two loops and interacting through a network of hydrogen bonds. This structure is consistent with the proposed mechanism for iPGM catalysis, with the two main steps in catalysis being a phosphatase reaction removing the phosphate from 2- or 3-phosphoglycerate, generating an enzyme-bound phosphoserine intermediate, followed by a phosphotransferase reaction as the phosphate is transferred from the enzyme back to the glycerate moiety. The structure also allowed the assignment of the function of the two domains of the enzyme, one of which participates in the phosphatase reaction and formation of the phosphoserine enzyme intermediate, with the other involved in the phosphotransferase reaction regenerating phosphoglycerate. Significant structural similarity has also been found between the active site of the iPGM domain catalyzing the phosphatase reaction and Escherichia coli alkaline phosphatase.

Amino Acid Sequence↗
PubMed2000

Investigations of the active site of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase using fluorescent labeled dolichyl-phosphate derivatives.

Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in the dolichol pathway that has been expressed as an active protein, solubilized and purified in large enough quantities for structural investigations. Earlier studies showed that the enzyme is closely associated with membranes of endoplasmic reticulum with unique lipid requirements for its maximal activity. This potential target of antibiotic therapy is now being investigated at the molecular level to establish information about the structure of the enzyme as well as determine the nature and properties of the enzyme-phospholipid interactions. In this paper, we have determined the activities of the fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular distances between amino acid residues near the active site and/or the fluorophores of the substrate derivatives using fluorescence energy resonance transfer. These results also show that the conserved consensus sequence is not required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the catalytic activity.

Amino Acid Sequence↗
PubMed2000

Crystal structure of a novel germination protease from spores of Bacillus megaterium: structural arrangement and zymogen activation.

The DNA in the core of spores of Bacillus species is saturated with a group of small, acid-soluble proteins (SASP) that protect DNA from a variety of harsh treatments and play a major role in spore resistance and long-term spore survival. During spore germination, SASPs are rapidly degraded to amino acids and this degradation is initiated by a sequence-specific protease called germination protease (GPR), which exhibits no obvious mechanistic or amino acid sequence similarity to any known class of proteases. GPR is synthesized during sporulation as an inactive tetrameric zymogen termed P(46), which later autoprocesses to a smaller form termed P(41), which is active only during spore germination. Here, we report the crystal structure of P(46) from Bacillus megaterium at 3.0 A resolution and the fact that P(46) monomer adopts a novel fold. The asymmetric unit contains two P(46) monomers and the functional tetramer is a dimer of dimers, with an approximately 9 A channel in the center of the tetramer. Analysis of the P(46) structure and site-directed mutagenesis studies have provided some insight into the mechanism of zymogen activation as well as the zymogen's lack of activity and the inactivity of P(41) in the mature spore.

Amino Acid Sequence↗
PubMed2000

Mechanism of hyaluronan binding and degradation: structure of Streptococcus pneumoniae hyaluronate lyase in complex with hyaluronic acid disaccharide at 1.7 A resolution.

Hyaluronic acid (HA) is an important constituent of the extracellular matrix; its bacterial degradation has been postulated to contribute to the spread of certain streptococci through tissue. Pneumococci and other streptococci produce hyaluronate lyase, an enzyme which depolymerizes HA, thus hyaluronate lyase might contribute directly to bacterial invasion. Although two different mechanisms for lyase action have been proposed, there was no crystallographic evidence to support those mechanisms. Here, we report the high-resolution crystal structure of Streptococcus pneumoniae hyaluronate lyase in the presence of HA disaccharide product, which ultimately provides the first crystallographic evidence for the binding of HA to hyaluronate lyase. This structural complex revealed a key interaction between the Streptococcus peneumoniae hyaluronate lyase protein and the product, and supports our previously proposed novel catalytic mechanism for HA degradation based on the native Streptococcus peneumoniae hyaluronate lyase structure. The information provided by this complex structure will likely be useful in the development of antimicrobial pharmaceutical agents.

Binding Sites↗
PubMed2000

Structure and mechanism of action of a novel phosphoglycerate mutase from Bacillus stearothermophilus.

Bacillus stearothermophilus phosphoglycerate mutase (PGM), which interconverts 2- and 3-phosphoglyceric acid (PGA), does not require 2,3-diphosphoglyceric acid for activity. However, this enzyme does have an absolute and specific requirement for Mn(2+) ions for catalysis. Here we report the crystal structure of this enzyme complexed with 3PGA and manganese ions to 1.9 A resolution; this is the first crystal structure of a diphosphoglycerate-independent PGM to be determined. This information, plus the location of the two bound Mn(2+) ions and the 3PGA have allowed formulation of a possible catalytic mechanism for this PGM. In this mechanism Mn(2+) ions facilitate the transfer of the substrate's phosphate group to Ser62 to form a phosphoserine intermediate. In the subsequent phosphotransferase part of the reaction, the phosphate group is transferred from Ser62 to the O2 or O3 positions of the reoriented glycerate to yield the PGA product. Site-directed mutagenesis studies were used to confirm our mechanism and the involvement of specific enzyme residues in Mn(2+) binding and catalysis.

Amino Acid Sequence↗
PubMed2000

Structural basis of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase.

Streptococcus pneumoniae hyaluronate lyase (spnHL) is a pathogenic bacterial spreading factor and cleaves hyaluronan, an important constituent of the extra- cellular matrix of connective tissues, through an enzymatic beta-elimination process, different from the hyaluronan degradation by hydrolases in animals. The mechanism of hyaluronan binding and degradation was proposed based on the 1.56 A resolution crystal structure, substrate modeling and mutagenesis studies on spnHL. Five mutants, R243V, N349A, H399A, Y408F and N580G, were constructed and their activities confirmed our mechanism hypothesis. The important roles of Tyr408, Asn349 and His399 in enzyme catalysis were proposed, explained and confirmed by mutant studies. The remaining weak enzymatic activity of the H399A mutant, the role of the free carboxylate group on the glucuronate residue, the enzymatic behavior on chondroitin and chondroitin sulfate, and the small activity increase in the N580G mutant were explained based on this mechanism. A possible function of the C-terminal beta-sheet domain is to modulate enzyme activity through binding to calcium ions.

Amino Acid Sequence↗
PubMed2000

Synthesis of dolichyl phosphate derivatives with fluorescent label at the omega-end of the chain, new tools to study protein glycosylation.

Derivatives of dolichyl phosphate (Dol-P) with 2-aminopyridine or 1-aminonaphtalene fluorophore groups at the omega-end of the chain were synthesized. These products serve as substrates for recombinant yeast Dol-P-mannose synthase. Fluorescence resonance energy transfer between a Trp residue of the enzyme and the 1-aminonaphtalene group of the Dol-P analogue was demonstrated.

1-Naphthylamine↗
PubMed2000

Production and characterization of the functional fragment of pneumococcal surface protein A.

Pneumococcal surface protein A (PspA) is present on the cell wall of Streptococcus pneumoniae pathogen and has an antigenetically variable N-terminal domain. This aminoterminal domain is essential for full pneumococcal virulence, and monoclonal antibodies raised against it protect mice against pneumococcal infections. We have cloned and expressed a 34-kDa N-terminal fragment of PspA in Escherichia coli in a soluble form using the T7 RNA polymerase pET-20b vector system. Nickel chelate affinity purification followed by size exclusion and anion exchange chromatography yielded large amounts of pure and homogeneous protein. Analytical ultracentrifugation sedimentation velocity band and boundary studies showed that the molecule was present in aqueous solutions in a monomeric form with an axial shape ratio of approximately 1:12, typical of fibrous proteins. Sequence analyses indicated an alpha-helical coiled-coil structure for this monomeric molecule with only few loop-type breaks in helicity. The mostly alpha-helical structure of this PspA construct was consistent with circular dichroism spectroscopy data. Based on the ultracentrifugation studies, the circular dichroism spectra, and the PspA's sequence analyses, two structural models for the amino-terminal part of the PspA molecule are proposed. The evident highly charged and polar character of the surface of the modeled structures suggests functional properties of PspA that are related to the prevention of S. pneumoniae interactions with the host complement system.

Amino Acid Sequence↗
PubMed2000

Structural characterization of penicillin-binding protein-related factor A (PrfA) from Bacillus species.

The prfA genes of Bacillus stearothermophilus and Bacillus subtilis are in an operon downstream of the ponA gene encoding penicillin-binding protein 1 (PBP1), a major enzyme involved in peptidoglycan synthesis. The specific function of the 23- to 24-kDa PrfA protein is unknown but this protein plays some role in nucleoid segregation and the functions of PrfA and PBP1 are interrelated. We overexpressed B. stearothermophilus and B. subtilis PrfA in Escherichia coli and purified the proteins to homogeneity by cation exchange and gel filtration chromatography. The protein is a monomer in solution, and circular dichroism spectroscopy revealed an abundance of beta-sheet secondary structure. Crystals of B. stearothermophilus PrfA were also obtained and diffracted X-rays to 1.8 A resolution.

Bacillus subtilis↗
PubMed2000

Structure and molecular mechanism of a functional form of pneumolysin: a cholesterol-dependent cytolysin from Streptococcus pneumoniae.

One of the key steps in understanding human disease arising from gram-positive bacteria lies in the mechanisms of the cholesterol-dependent cytolysins (CDCs). Pneumolysin (PLY), a CDC from Streptococcus pneumoniae, is of special importance due to the severe impacts of pneumococcal infections on mortality and morbidity worldwide. We have overexpressed, purified, and characterized PLY in its fully functional complex form with the enzyme bound to its receptor activator on target cells, cholesterol. The circular dichroism studies of PLY in solution with an excess of cholesterol show a change in the far UV spectrum consistent with a decrease in the beta-sheet and an increase in the random coil structures of the enzyme. Pore formation in membranes leading to cell lysis is the functional target for this cytolysin. The sedimentation velocity and equilibrium analyses of the cholesterol-bound enzyme show hydrodynamic properties different from those of the cholesterol-free form. The soluble form of the cholesterol-free enzyme exists in solution as a mixture of monomers and dimers, whereas the cholesterol-bound form exists only as a monomer. A mechanism of formation of PLY pores in the lipid bilayer of the target cells is discussed.

Bacterial Proteins↗