Cynthia V. Stauffacher

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 ‐induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.

Enterococcus faecalis Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis

Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, DeltaH(a) and K(m) values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K(i) for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme.

Crystal Structure of a Human Low Molecular Weight Phosphotyrosyl Phosphatase IMPLICATIONS FOR SUBSTRATE SPECIFICITY

The low molecular weight phosphotyrosine phosphatases (PTPases) constitute a distinctive class of phosphotyrosine phosphatases that is widely distributed among vertebrate and invertebrate organisms. In vertebrates, two isoenzymes of these low molecular weight PTPases are commonly expressed. The two human isoenzymes, HCPTPA and HCPTPB, differ in an alternatively spliced sequence (residues 40–73) referred to as the variable loop, resulting in isoenzymes that have different substrate specificities and inhibitor/activator responses. We present here the x-ray crystallographic structure of a human low molecular weight PTPase solved by molecular replacement to 2.2 Å. The structure of human low molecular weight PTPase is compared with a structure representing the other isoenzyme in this PTPase class, in each case with a sulfonate inhibitor bound to the active site. Possible aromatic residue interactions with the phosphotyrosine substrate are proposed from an examination of the binding site of the inhibitors. Differences are observed in the variable loop region, which forms one wall and the floor of a long crevice leading from the active-site loop. A set of residues lying along this crevice (amino acids 49, 50, and 53) is suggested to be responsible for differences in substrate specificity in these two enzymes.

Essentiality, Expression, and Characterization of the Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase of Staphylococcus aureus

Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus. In this study we showed through genetic disruption experiments that mvaA, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S. aureus. Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The mvaA gene was cloned from S. aureus DNA and expressed with an N-terminal His tag in Escherichia coli. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the S. aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a K(i) of 320 microM, over 10(4) higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.

Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductases

The biosynthesis of isopentenyl diphosphate (isopentenyl pyrophosphate), the precursor of isoprenoids in all forms of life, occurs by two distinct metabolic pathways, the mevalonate pathway (Fig. 1) and the glyceraldehyde 3-phosphate/pyruvate pathway, often termed the nonmevalonate pathway (17). Whereas many gram-negative bacteria employ the nonmevalonate pathway (26), humans, other eukaryotes, archaea, grampositive cocci, and the spirochete Borrelia burgdorferi utilize the enzymes and intermediates of the mevalonate pathway (15, 16, 20, 21, 26). This review addresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the catalyst for the ratelimiting reaction of the mevalonate pathway for isopentenyl diphosphate biosynthesis. HMG-CoA reductase catalyzes the reductive deacylation of (S)-HMG-CoA to (R)-mevalonate: The reaction proceeds in three stages, the first and third of which are reductive, and it involves successive formation of enzyme-bound mevaldyl-CoA and mevaldehyde. While mevaldehyde is not released during the course of the reaction, HMG-CoA reductase catalyzes two reactions of free mevaldehyde. Reaction 2 resembles the third stage, and reaction 3 resembles the reverse of stages 1 and 2 of the overall reaction 1.

Enterococcus faecalis 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis

Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni(2+)-agarose to apparent homogeneity and a specific activity of 10 micromol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s(20,w), 5.3). Optimal activity occurred in 2.0 mM MgCl(2) at 37(o)C. The DeltaH(a) was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK(a) of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 +/- 0.2 and that of covalent acetylation was 0.60 +/- 0.02. The K(m) for the hydrolysis of acetyl-CoA was 10 microM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis.

Subtype-specific interactions of type C staphylococcal enterotoxins with the T-cell receptor

The goal of this study was to investigate the molecular interaction between superantigens and the T‐cell receptor (TCR). Using a quantitative polymerase chain reaction (PCR) to assess T‐cell proliferation profiles, we found that SEB, SEC1, SEC2 and SEC3 expanded human T cells bearing Vβ3, Vβ12, Vβ13.2, Vβ14, Vβ15, Vβ17 and Vβ20. SEC2 and SEC3 have the additional ability to expand T cells bearing Vβ13.1, and their expansion of Vβ3 was markedly reduced compared to SEB and SEC1. Based on the activity of SEC1 mutants containing single amino acid substitutions, we concluded that the differential abilities of these native toxins to stimulate Vβ3 and Vβ13.1 was determined by the residue in position 26, located in the base of the SEC α3 cavity. The SEC1 mutant, in which Val in position 26 was substituted with the analogous SEC2/SEC3 residue (Tyr), generated a Vβ expansion profile that was indistinguishable from those generated by SEC2 and SEC3. Using these findings, the co‐ordinates of a recently reported murine TCR β‐chain crystal structure, and other documented information, we propose a compatible molecular model for the interaction of SEC3 with the T‐cell receptor. In this model complex, the complementarity‐determining regions (CDRs) 1 and 2 and the hypervariable loop 4 of the Vβ element contact SEC3 predominantly through residues in the α3 cavity of the toxin. CDR3 of the β chain is not involved in any toxin contacts. The proposed model not only includes contacts identified in previous mutagenesis studies, but is also consistent with the ability of tyrosine and valine in position 26 to differentially affect the expansion of Vβs 3 and 13.1 by the SEC superantigens.

Molecular structure of staphylococcus and streptococcus superantigens

Staphylococcus aureus and streptococci, notably those belonging to group A, make up a large family of true exotoxins referred to as pyrogenic toxin superantigens. These toxins cause toxic shock-like syndromes and have been implicated in several allergic and autoimmune diseases. Included within this group of proteins are the staphylococcal enterotoxins, designated serotypes A, B, Cn, D, E, and G; two forms of toxic shock syndrome toxin-1 also made byStaphylococcus aureus; the group A streptococcal pyrogenic exotoxins, serotypes A, B, and C; and recently described toxins associated with groups B, C, F, and G streptococci. The nucleotide sequences of the genes for all of the toxins except those from the groups B, C, F, and G streptococcal strains have been sequenced. The sequencing studies indicate that staphylococcal enterotoxins B and C and streptococcal pyrogenic exotoxin A share highly significant sequence similarity; staphylococcal enterotoxins A, D, and E share highly significant sequence similarity; and toxic shock syndrome toxin-1 and streptococcal pyrogenic exotoxin B and C share little, if any, sequence similarity with any of the toxins. Despite the dissimilarities seen in primary amino acid sequence among some members of the toxin family, it was hypothesized that there was likely to be significant three-dimensional structure similarity among all the toxins. The three-dimensional structures of three of the pyrogenic toxin superantigens have been determined recently. The structural features of two of these, toxic shock syndrome toxin-1 and enterotoxin C3, are presented. Toxic shock syndrome-1 exists as a protein with two major domains, referred to as A and B. The molecule begins with a short N-terminalα-helix that then leads into a clawshaped structure in domain B that is made up ofβ strands. Domain B is connected to domain A by a central diagonalα-helix of amino acids which are important in both the superantigenic and the lethal activities of the toxin. Finally, domain A contains a wall ofβ strands and the C terminus of the molecule. The small N-terminalα-helix and the twoβ sheet structures (claw and wall) form part of a deep groove on the back side of the toxin that contains the centralα-helix. Staphylococcal enterotoxin C3 differs somewhat from toxic shock syndrome toxin-1: it has an elongated N terminus that folds over domain A, anα-helix at the base of domain B, a cysteine loop structure above the claw structure in domain B of toxic shock syndrome toxin-1, and a second centralα-helix.

Crystal Structure of the Human B-form Low Molecular Weight Phosphotyrosyl Phosphatase at 1.6-Å Resolution

The crystal structure of HPTP-B, a human isoenzyme of the low molecular weight phosphotyrosyl phosphatase (LMW PTPase) is reported here at a resolution of 1.6 Å. This high resolution structure of the second human LMW PTPase isoenzyme provides the opportunity to examine the structural basis of different substrate and inhibitor/activator responses. The crystal packing of HPTP-B positions a normally surface-exposed arginine in a position equivalent to the tyrosyl substrate. A comparison of all deposited crystallographic coordinates of these PTPases reveals three atomic positions within the active site cavity occupied by hydrogen bond donor or acceptor atoms on bound molecules, suggesting useful design elements for synthetic inhibitors. A selection of inhibitor and activator molecules as well as small molecule and peptide substrates were tested against each human isoenzyme. These results along with the crystal packing seen in HPTP-B suggest relevant sequence elements in the currently unknown target sequence.

The Structure of Cowpea Mosaic Virus at 3.5 Å Resolution

There are nine groups of icosahedral, positive stranded RNA plant viruses (Francki et al., 1985) established on the basis of immunological relationships, genomic size, the number of RNA molecules composing the genome and the stability of the capsid. Eight of these groups are remarkably similar in both structural and functional properties while one group, the comoviruses, is quite different from the others (Goldbach and vanKammen, 1985). Cowpea mosaic virus (CPMV), the type member of the comovirus group, has only two properties in common with any of the other plant virus groups. The genome of CPMV is bipartite and a small protein (VpG) is linked to the 5′ terminus of each RNA molecule (Bruening, 1977; Daubert et al., 1979). In all its other properties CPMV is unique among the plant viruses. The RNA molecules of CPMV are polyadenylated at their 3′ termini while the genomes of the other virus groups are not. CPMV proteins are generated by the processing of two polyproteins, produced by translation of single open reading frames on each RNA molecule. The proteases that cleave the polyproteins are virally encoded and are initially part of the translation product of the larger of the two RNA molecules. The other plant virus groups utilize subgenomic RNA molecules as the messengers for protein synthesis with no posttranslational processing. The CPMV genome codes for a total of eight proteins, three (including the capsid proteins) derived from the small RNA molecule, and five from the large RNA.