Mark J. Jedrzejas

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 extracellular matrix of connective tissues, through an enzymatic β‐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 Å 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 β‐sheet domain is to modulate enzyme activity through binding to calcium ions.

Amidase domains from bacterial and phage autolysins define a family of γ-d,l-glutamate-specific amidohydrolases

Several phage-encoded peptidoglycan hydrolases have been found to share a conserved amidase domain with a variety of bacterial autolysins (N-acetylmuramoyl-L-alanine amidases), bacterial and eukaryotic glutathionylspermidine amidases, gamma-D-glutamyl-L-diamino acid endopeptidase and NLP/P60 family proteins. All these proteins contain conserved cysteine and histidine residues and hydrolyze gamma-glutamyl-containing substrates. These cysteine residues have been shown to be essential for activity of several of these amidases and their thiol groups apparently function as the nucleophiles in the catalytic mechanisms of all enzymes containing this domain. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) superfamily includes a variety of previously uncharacterized proteins, including the tail assembly protein K of phage lambda. Some members of this superfamily are important surface antigens in pathogenic bacteria and might represent drug and/or vaccine targets.

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. Proteins 2001;45:318–324.

Structure, function, and evolution of phosphoglycerate mutases: comparison with fructose-2,6-bisphosphatase, acid phosphatase, and alkaline phosphatase

2. Cofactor dependent Saccharomyces cerevisiae phosphoglycerate mutase . . . . . . . . . . . . . . . . 265 2.1. Structural aspects of Saccharomyces cerevisiae phosphoglycerate mutase . . . . . . . . . . 265 2.2. Mechanism of catalysis of the S. cerevisiae dPGM . . . . . . . . . . . . . . . . . . . . . . . . . . 270 2.3. Structural comparison of dPGM with other enzymes . . . . . . . . . . . . . . . . . . . . . . . . 270 2.3.1. Structural comparison of S. cerevisiae dPGM with 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 2.3.2. Structural comparison of S. cerevisiae dPGM with acid phosphatase . . . . . . . 272 2.3.3. Overall comparison of active sites of dPGM, Fru26P2ase, and AcPase . . . . . 273

Mechanism of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase - Structures of complexes with the substrate

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniaehyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-Å resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn349, His399, and Tyr408, is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the α- and β-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme.

Structure of a protein–DNA complex essential for DNA protection in spores of Bacillus species

The DNA-binding α/β-type small acid-soluble proteins (SASPs) are a major factor in the resistance and long-term survival of spores of Bacillus species by protecting spore DNA against damage due to desiccation, heat, toxic chemicals, enzymes, and UV radiation. We now report the crystal structure at 2.1 Å resolution of an α/β-type SASP bound to a 10-bp DNA duplex. In the complex, the α/β-type SASP adopt a helix–turn–helix motif, interact with DNA through minor groove contacts, bind to ≈6 bp of DNA as a dimer, and the DNA is in an A-B type conformation. The structure of the complex provides important insights into the molecular details of both DNA and α/β-type SASP protection in the complex and thus also in spores.

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 Mn2+ ions for catalysis. Here we report the crystal structure of this enzyme complexed with 3PGA and manganese ions to 1.9 Å resolution; this is the first crystal structure of a diphosphoglycerate‐independent PGM to be determined. This information, plus the location of the two bound Mn2+ ions and the 3PGA have allowed formulation of a possible catalytic mechanism for this PGM. In this mechanism Mn2+ ions facilitate the transfer of the substrates 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 Mn2+ binding and catalysis.

Carbohydrate Polymers at the Center of Life’s Origins: The Importance of Molecular Processivity

2. Current Polymeric Glycans 5070 2.1. Structural Polysaccharides 5071 2.1.1. Cellulose 5071 2.1.2. CT 5071 2.1.3. Bacterial Cell Wall and Surface Glycans 5071 2.1.4. Consequences of Structural Polysaccharide Evolution 5071 2.2. Storage Polysaccharides 5071 2.2.1. Starch 5071 2.2.2. Glycogen 5071 2.2.3. Pectins 5072 2.2.4. Alginate 5072 2.3. ECM and GAGs 5072 2.3.1. Heparin and HS 5072 2.3.2. Ch and ChSs 5072 2.3.3. DS 5073 2.3.4. KS 5073 2.3.5. HA 5073 2.4. Polysaccharides of Other Glycoproteins 5073 2.4.1. Branched Polysaccharides of Mucins 5073 2.4.2. Oligosaccharides of Heterogeneous Glycoproteins 5073

Structural and Functional Comparison of Polysaccharide-Degrading Enzymes

Sugar molecules as well as enzymes degrading them are ubiquitously present in physiological systems, especially for vertebrates. Polysaccharides have at least two aspects to their function, one due to their mechanical properties and the second one involves multiple regulatory processes or interactions between molecules, cells, or extracellular space. Various bacteria exert exogenous pressures on their host organism to diversity glycans and their structures in order for the host organism to evade the destructive function of such microbes. Many bacterial organism produce glycan-degrading enzymes in order to facilitate their invasion of host tissues. Such polysaccharide degrading enzymes utilize mainly two modes of polysaccharide-degradation, a hydrolysis and a β-elimination process. The three-dimensional structures of several of these enzymes have been elucidated recently using X-ray crystallography. There are many common structural motifs among these enzymes, mainly the presence of an elongated cleft transversing these molecules which functions as a polysaccharide substrate binding site as well as the catalytic site for these enzymes. The detailed structural information obtained about these enzymes allowed formulation of proposed mechanisms of their action. The polysaccharide lyases utilize a proton acceptance and donation mechanism (PAD), whereas polysaccharide hydrolases use a direct double displacement (DD) mechanism to degrade their substrates.

Analysis of structure and function of putative surface-exposed proteins encoded in the Streptococcus pneumoniae genome: a bioinformatics-based approach to vaccine and drug design.

Streptococcus pneumoniae is the most common cause of fatal community-acquired pneumonia, middle ear infection, and meningitis. The prevention and treatment of this infection have become a top priority for the medical-scientific community. The present polysaccharide-based vaccine used to immunize susceptible hosts is only ∼60% effective and is ineffective in children younger than 2 years of age. The new conjugate vaccine, based on the engineered diphtheria toxin coupled to polysaccharide antigens, is approved only for use in children under 2 years of age to treat invasive disease. While penicillin is the drug of choice to treat infections secondary to S. pneumoniae, increasing numbers of bacterial strains are resistant to penicillin as well as to broad spectrum antibiotics such as vancomycin. Thus, there is a need to identify new strategies to prevent and treat diseases caused by to S. pneumoniae. In this article, we summarize the utilization of the recently available S. pneumoniae genomic information in order to identify and characterize novel proteins likely located on the surface of this Gram-positive pathogenic bacterium. Because only a limited number of surface proteins of S. pneumoniae have been characterized to date, this information provides new insights into the pathogenesis of this organism as well as highlights possible avenues for its treatment and/or prevention in the future. The review is divided into two sections. First, we briefly summarize current information about known surface-exposed proteins of S. pneumoniae. This is followed by the illustration of procedures for the identification of new putative surface-exposed proteins. These have signal peptides required for their extra-cytoplasmic transport and/or additional signature sequences. Some of these will be S. pneumoniae virulence factors. The signature sequences we have chosen are those leading to protein binding to choline present on the bacterial surface, attachment to peptidoglycan of the cell wall, or anchoring to lipids of the cytoplasmic membrane. All these signatures are indicative of binding of proteins to the surface of this organism. Secondly, we illustrate the application of bioinformatics and modeling tools to these selected proteins in order to provide information about their likely functions and preliminary three-dimensional structure models. The focal point of the analysis of these proteins, their sequences, and structures is the evaluation of their antigenic properties and possible roles in pathogenicity. The information obtained from the genome analysis will be instrumental in the development of a more effective prophylactic and/or therapeutic agents to prevent and to treat infections due to S. pneumoniae.