Characterization of a thiamin diphosphate-dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae

The product of the ARO10 gene from Saccharomyces cerevisiae was initially identified as a thiamine diphosphate-dependent phenylpyruvate decarboxylase with a broad substrate specificity. It was suggested that the enzyme could be responsible for the catabolism of aromatic and branched-chain amino acids, as well as methionine. In the present study, we report the overexpression of the ARO10 gene product in Escherichia coli and the first detailed in vitro characterization of this enzyme. The enzyme is shown to be an efficient aromatic 2-keto acid decarboxylase, consistent with it playing a major in vivo role in phenylalanine, tryptophan and possibly also tyrosine catabolism. However, its substrate spectrum suggests that it is unlikely to play any significant role in the catabolism of the branched-chain amino acids or of methionine. A homology model was used to identify residues likely to be involved in substrate specificity. Site-directed mutagenesis on those residues confirmed previous studies indicating that mutation of single residues is unlikely to produce the immediate conversion of an aromatic into an aliphatic 2-keto acid decarboxylase. In addition, the enzyme was compared with the phenylpyruvate decarboxylase from Azospirillum brasilense and the indolepyruvate decarboxylase from Enterobacter cloacae. We show that the properties of the two phenylpyruvate decarboxylases are similar in some respects yet quite different in others, and that the properties of both are distinct from those of the indolepyruvate decarboxylase. Finally, we demonstrate that it is unlikely that replacement of a glutamic acid by leucine leads to discrimination between phenylpyruvate and indolepyruvate, although, in this case, it did lead to unexpected allosteric activation.     

Loop movement and catalysis in creatine kinase

Recently the crystal structure of creatine kinase from Torpedocalifornica was determined to 2.1 A. The dimeric structure revealed two different forms in the unit cell: one monomer was bound to a substrate, MgADP, and the other monomer was bound to a transition-state analogue complex composed of MgADP, nitrate and creatine. The most striking difference between the structures is the movement of two loops (comprising residues 60-70 and residues 323-333) into the active site in the transition state structure. This loop movement effectively occludes the active site from solvent, and the loops appear to be locked into place by a salt bridge formed between His66 and Asp326. His66 is of particular interest as it is located within a PGHP motif conserved in all creatine kinases but not found in other guanidino kinases. We have carried out alanine-scanning mutagenesis of each of the residues in the PGHP motif and determined that only the His66 plays a significant role in the creatine kinase reaction. Although neither residue interacts directly with the substrate, the interaction His66 and Asp326 appears to be important in providing the precise alignment of substrates necessary for phosphoryl group transfer. Finally, it is clear that neither His66 nor Asp326 are responsible for the pKs observed in the pH-rate profile for HMCK.     

Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active

The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide-linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design.     

Mandelamide hydrolase from Pseudomonas putida: characterization of a new member of the amidase signature family

A recently discovered enzyme in the mandelate pathway of Pseudomonas putida, mandelamide hydrolase (MAH), catalyzes the hydrolysis of mandelamide to mandelic acid and ammonia. Sequence analysis suggests that MAH is a member of the amidase signature family, which is widespread in nature and contains a novel Ser-cis-Ser-Lys catalytic triad. Here we report the expression in Escherichia coli, purification, and characterization of both wild-type and His(6)-tagged MAH. The recombinant enzyme was stable, exhibited a pH optimum of 7.8, and was able to hydrolyze both enantiomers of mandelamide with little enantiospecificity. The His-tagged variant showed no significant change in kinetic constants. Phenylacetamide was found to be the best substrate, with changes in chain length or replacement of the phenyl group producing greatly decreased values of k(cat)/K(m). As with another member of this family, fatty acid amide hydrolase, MAH has the uncommon ability to hydrolyze esters and amides at similar rates. MAH is even more unusual in that it will only hydrolyze esters and amides with little steric bulk. Ethyl and larger esters and N-ethyl and larger amides are not substrates, suggesting that the MAH active site is very sterically hindered. Mutation of each residue in the putative catalytic triad to alanine resulted in total loss of activity for S204A and K100A, while S180A exhibited a 1500-fold decrease in k(cat) and significant increases in K(m) values. Overall, the MAH data are similar to those of fatty acid amide hydrolase and support the suggestion that there are two distinct subgroups within the amidase signature family.     

Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase

Thiamin diphosphate-dependent decarboxylases catalyze the non-oxidative decarboxylation of 2-keto carboxylic acids. Although they display relatively low sequence similarity, and broadly different range of substrates, these enzymes show a common homotetrameric structure. Here we describe a kinetic characterization of the substrate spectrum of a recently identified member of this class, the branched chain 2-keto acid decarboxylase (KdcA) from Lactococcus lactis. In order to understand the structural basis for KdcA substrate recognition we developed a homology model of its structure. Ser286, Phe381, Val461 and Met358 were identified as residues that appeared to shape the substrate binding pocket. Subsequently, site-directed mutagenesis was carried out on these residues with a view to converting KdcA into a pyruvate decarboxylase. The results show that the mutations all lowered the Km value for pyruvate and both the S286Y and F381W variants also had greatly increased values of k(cat) with pyruvate as a substrate.     

Exploring the role of the active site cysteine in human muscle creatine kinase

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.     

Olfactomedin 4 Inhibits Cathepsin C-Mediated Protease Activities, Thereby Modulating Neutrophil Killing of Staphylococcus aureus and Escherichia coli in Mice

Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4(-/-) mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4(-/-) mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4(-/-) mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4(-/-) neutrophils. The bacterial killing and clearance capabilities observed in OLFM4(-/-) mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.     

Host-plant species conservatism and ecology of a parasitoid fig wasp genus (chalcidoidea; sycoryctinae; arachonia)

Parasitoid diversity in terrestrial ecosystems is enormous. However, ecological processes underpinning their evolutionary diversification in association with other trophic groups are still unclear. Specialisation and interdependencies among chalcid wasps that reproduce on Ficus presents an opportunity to investigate the ecology of a multi-trophic system that includes parasitoids. Here we estimate the host-plant species specificity of a parasitoid fig wasp genus that attacks the galls of non-pollinating pteromalid and pollinating agaonid fig wasps. We discuss the interactions between parasitoids and the Ficus species present in a forest patch of Uganda in context with populations in Southern Africa. Haplotype networks are inferred to examine intraspecific mitochondrial DNA divergences and phylogenetic approaches used to infer putative species relationships. Taxonomic appraisal and putative species delimitation by molecular and morphological techniques are compared. Results demonstrate that a parasitoid fig wasp population is able to reproduce on at least four Ficus species present in a patch. This suggests that parasitoid fig wasps have relatively broad host-Ficus species ranges compared to fig wasps that oviposit internally. Parasitoid fig wasps did not recruit on all available host plants present in the forest census area and suggests an important ecological consequence in mitigating fitness trade-offs between pollinator and Ficus reproduction. The extent to which parasitoid fig wasps exert influence on the pollination mutualism must consider the fitness consequences imposed by the ability to interact with phenotypes of multiple Ficus and fig wasps species, but not equally across space and time.     

Comparison of proteins expressed on secretory vesicle membranes and plasma membranes of human neutrophils

Secretory vesicles are neutrophil intracellular storage granules formed by endocytosis. Understanding the functional consequences of secretory vesicle exocytosis requires knowledge of their membrane proteins. The current study was designed to use proteomic technologies to develop a more complete catalog of secretory vesicle membrane proteins and to compare the proteomes of secretory vesicle and plasma membranes. A total of 1118 proteins were identified, 573 (51%) were present only in plasma membrane-enriched fractions, 418 (37%) only in secretory vesicle-enriched membrane fractions, and 127 (11%) in both fractions. Gene Ontology categorized 373 of these proteins as integral membrane proteins. Proteins typically associated with other intracellular organelles, including nuclei, mitochondria, and ribosomes, were identified in both membrane fractions. Ingenuity Pathway Knowledge Base analysis determined that the majority of canonical and functional pathways were significantly associated with proteins from both plasma membrane-enriched and secretory vesicle-enriched fractions. There were, however, some canonical signaling pathways that involved proteins only from plasma membranes or secretory vesicles. In conclusion, a number of proteins were identified that may elucidate mechanisms and functional consequences of secretory vesicle exocytosis. The small number of common proteins suggests that the hypothesis that secretory vesicles are formed from plasma membranes by endocytosis requires more critical evaluation.     

The cloning and over-expression of PABA synthase in E. coli

Both the genes encoding E. coli p-aminobenzoic acid synthase have been cloned and an overproducing strain has been obtained. The partial purification of the large subunit is described. The kinetic properties of the cloned enzyme, while similar to those reported for the B. subtilis enzyme, show some differences to those reported for the S. griseus enzyme.