Linda A. Fothergill-Gilmore

Crystal Structure of a Thermostable Lipase from Bacillus stearothermophilus P1

We describe the first lipase structure from a thermophilic organism. It shares less than 20% amino acid sequence identity with other lipases for which there are crystal structures, and shows significant insertions compared with the typical alpha/beta hydrolase canonical fold. The structure contains a zinc-binding site which is unique among all lipases with known structures, and which may play a role in enhancing thermal stability. Zinc binding is mediated by two histidine and two aspartic acid residues. These residues are present in comparable positions in the sequences of certain lipases for which there is as yet no crystal structural information, such as those from Staphylococcal species and Arabidopsis thaliana. The structure of Bacillus stearothermophilus P1 lipase provides a template for other thermostable lipases, and offers insight into mechanisms used to enhance thermal stability which may be of commercial value in engineering lipases for industrial uses.

M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation

We show that the M2 isoform of pyruvate kinase (M2PYK) exists in equilibrium between monomers and tetramers regulated by allosteric binding of naturally occurring small-molecule metabolites. Phenylalanine stabilizes an inactive T-state tetrameric conformer and inhibits M2PYK with an IC50 value of 0.24 mM, whereas thyroid hormone (triiodo-l-thyronine, T3) stabilizes an inactive monomeric form of M2PYK with an IC50 of 78 nM. The allosteric activator fructose-1,6-bisphosphate [F16BP, AC50 (concentration that gives 50% activation) of 7 μM] shifts the equilibrium to the tetrameric active R-state, which has a similar activity to that of the constitutively fully active isoform M1PYK. Proliferation assays using HCT-116 cells showed that addition of inhibitors phenylalanine and T3 both increased cell proliferation, whereas addition of the activator F16BP reduced proliferation. F16BP abrogates the inhibitory effect of both phenylalanine and T3, highlighting a dominant role of M2PYK allosteric activation in the regulation of cancer proliferation. X-ray structures show constitutively fully active M1PYK and F16BP-bound M2PYK in an R-state conformation with a lysine at the dimer-interface acting as a peg in a hole, locking the active tetramer conformation. Binding of phenylalanine in an allosteric pocket induces a 13° rotation of the protomers, destroying the peg-in-hole R-state interface. This distinct T-state tetramer is stabilized by flipped out Trp/Arg side chains that stack across the dimer interface. X-ray structures and biophysical binding data of M2PYK complexes explain how, at a molecular level, fluctuations in concentrations of amino acids, thyroid hormone, and glucose metabolites switch M2PYK on and off to provide the cell with a nutrient sensing and growth signaling mechanism.

Chitinases from Vibrio: activity screening and purification of chiA from Vibrio carchariae

Fourteen species of Vibrio were screened for chitin‐induced chitinase activity in culture medium. V. carchariae, V. alginolyticus 283 and V. campbellii showed high levels of activity. Screening on agar plates containing swollen chitin showed high levels of chitinase activity by the same three species, and also by V. fischeri and V. alginolyticus 284. An affinity purification procedure was developed for the chitinase from V. carchariae. The purified chitinase was active as a monomer with Mr 63 000–66 000, and displayed activity toward polymeric chitin from acetylated chitosan or from crab shells. N‐terminal sequence analysis and immunological cross‐reactivity confirmed that the enzyme belongs to the group A/chiA family of bacterial chitinases.

The trypanocidal drug suramin and other trypan blue mimetics are inhibitors of pyruvate kinases and bind to the adenosine site

Ehrlichs pioneering chemotherapeutic experiments published in 1904 (Ehrlich, P., and Shiga, K. (1904) Berlin Klin. Wochenschrift 20, 329–362) described the efficacy of a series of dye molecules including trypan blue and trypan red to eliminate trypanosome infections in mice. The molecular structures of the dyes provided a starting point for the synthesis of suramin, which was developed and used as a trypanocidal drug in 1916 and is still in clinical use. Despite the biological importance of these dye-like molecules, the mode of action on trypanosomes has remained elusive. Here we present crystal structures of suramin and three related dyes in complex with pyruvate kinases from Leishmania mexicana or from Trypanosoma cruzi. The phenyl sulfonate groups of all four molecules (suramin, Ponceau S, acid blue 80, and benzothiazole-2,5-disulfonic acid) bind in the position of ADP/ATP at the active sites of the pyruvate kinases (PYKs). The binding positions in the two different trypanosomatid PYKs are nearly identical. We show that suramin competitively inhibits PYKs from humans (muscle, tumor, and liver isoenzymes, Ki = 1.1–17 μm), T. cruzi (Ki = 108 μm), and L. mexicana (Ki = 116 μm), all of which have similar active sites. Synergistic effects were observed when examining suramin inhibition in the presence of an allosteric effector molecule, whereby IC50 values decreased up to 2-fold for both trypanosomatid and human PYKs. These kinetic and structural analyses provide insight into the promiscuous inhibition observed for suramin and into the mode of action of the dye-like molecules used in Ehrlichs original experiments.

Allosteric Mechanism of Pyruvate Kinase from Leishmania mexicana Uses a Rock and Lock Model

Allosteric regulation provides a rate management system for enzymes involved in many cellular processes. Ligand-controlled regulation is easily recognizable, but the underlying molecular mechanisms have remained elusive. We have obtained the first complete series of allosteric structures, in all possible ligated states, for the tetrameric enzyme, pyruvate kinase, from Leishmania mexicana. The transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6° rigid body rocking motion of the A- and C-domain cores in each of the four subunits. However, formation of the R-state in this way is only part of the mechanism; eight essential salt bridge locks that form across the C-C interface provide tetramer rigidity with a coupled 7-fold increase in rate. The results presented here illustrate how conformational changes coupled with effector binding correlate with loss of flexibility and increase in thermal stability providing a general mechanism for allosteric control.

Design, synthesis and trypanocidal activity of lead compounds based on inhibitors of parasite glycolysis

The glycolytic pathway has been considered a potential drug target against the parasitic protozoan species of Trypanosoma and Leishmania. We report the design and the synthesis of inhibitors targeted against Trypanosoma brucei phosphofructokinase (PFK) and Leishmania mexicana pyruvate kinase (PyK). Stepwise library synthesis and inhibitor design from a rational starting point identified furanose sugar amino amides as a novel class of inhibitors for both enzymes with IC(50) values of 23microM and 26microM against PFK and PyK, respectively. Trypanocidal activity also showed potency in the low micromolar range and confirms these inhibitors as promising candidates for the development towards the design of anti-trypanosomal drugs.

The role of the C-terminal region in phosphoglycerate mutase

Removal of the C-terminal seven residues from phosphoglycerate mutase from Saccharomyces cerevisiae by limited proteolysis is associated with loss of mutase activity, but no change in phosphatase activity. The presence of the cofactor 2, 3-bisphosphoglycerate, or of the cofactor and substrate 3-phosphoglycerate together, confers protection against proteolysis. The substrate alone offers no protection. Replacement of either or both of the two lysines at the C-terminus by glycines has only limited effects on the kinetic properties of phosphoglycerate mutase, indicating that these residues are unlikely to be involved in crucial electrostatic interactions with the substrate, intermediate or product in the reaction. However, the double-mutant form of the enzyme is more sensitive to proteolysis and is no longer protected against proteolysis by the presence of cofactor. The proteolysed wild-type and two of the mutated forms of the enzyme show a reduced response to 2-phosphoglycollate, which enhances the instability of the phospho form of the native enzyme. The phosphoglycerate mutase from Schizosaccharomyces pombe, which lacks the analogous C-terminal tail, has an inherently lower mutase activity and is also less responsive to stimulation by 2-phosphoglycollate. It is proposed that the C-terminal region of phosphoglycerate mutase helps to maintain the enzyme in its active phosphorylated form and assists in the retention of the bisphosphoglycerate intermediate at the active site. However, its role seems not to be to contribute directly to ligand binding, but rather to exert indirect effects on the transfer of the phospho group between substrate, enzyme, intermediate and product.

TRAMP (Tyrosine Rich Acidic Matrix Protein), a Protein that Co-purifies with Lysyl Oxidase from Porcine Skin: Identification of TRAMP as the dermatan sulphate proteoglycan-associated 22K extracellular matrix protein

A protein (M(r)24 K) that co-purifies with porcine skin lysyl oxidase (M(r)34 K) has been isolated and characterised. Five variants of the 24 K protein were identified by Mono Q ion-exchange FPLC, as were four variants of lysyl oxidase. Amino acid analysis and partial sequencing revealed near identity of a 36-residue CNBr peptide from porcine skin lysyl oxidase to corresponding regions of the putative lysyl oxidase precursor derived from rat and human cDNA. The 24 K protein was found to be unrelated to lysyl oxidase, but comparison with a protein sequence database showed it to be the same as a recently described protein from bovine skin that is associated with dermatan sulphate proteoglycans. The 24 K protein is relatively rich in tyrosine, and isoelectric focussing shows it to be acidic, with pIs in the range 4.1 to 4.4. In view of these properties, we propose the name TRAMP (Tyrosine Rich Acidic Matrix Protein) to identify this protein. Though TRAMP appears not to be glycosylated, several experiments indicate the presence of sulphotyrosine residues. When assayed using an elastin substrate, the activity of lysyl oxidase is unaffected by TRAMP.

The yeast pyruvate kinase gene does not contain a string of non‐ preferred codons: Revised nucleotide sequence

The sequence of the gene encoding pyruvate kinase from Saccharomyces cerevisiae was re‐determined because of failures with oligonucleotide‐directed mutagenesis experiments involving a region thought to contain a string of five contiguous non‐preferred codons. This region was found to be difficult to sequence and was shown to have three extra bases when compared with the published sequence [(1983) J. Biol. Chem. 258, 2193–2201]. The revised sequence demonstrates that the yeast pyruvate kinase gene does not have a cluster of non‐preferred codons, and that it therefore is not an example of the class of genes which possibly exhibit translational control by the presence of non‐preferred codons.

Sequence of the gene encoding phosphoglycerate mutase from Saccharomyces cerevisiae

The gene encoding yeast phosphoglycerate mutase was isolated, and its sequence was determined. The gene specifies a protein of 246 amino acids, and contains no introns. The sequence shows a strong codon bias. The upstream untranslated portion of the gene contains a CT‐rich block such as is found in many highly expressed yeast genes, but does not have the associated CAAG sequence.