Gene sequences encoding ribosome-inactivating proteins from soapwort (Saponaria officinalis L.)

Ribosome-inactivating proteins (RIPs) are found in a wide variety of plant species. They possess an RNA N-glycosidase activity whereby the removal of a specific adenine residue from 28 S RNA renders a eukaryotic ribosome inactive. Type II RIPS contain both an active polypeptide and a sugar-binding polypeptide. Type I RIPs are composed of a single polypeptide functionally homologous to the active type II polypeptide. This thesis describes studies of the gene sequences of RIPs representative of each class: Ricin, a type II RIP from the castor oil plant (Ricinus communis h.), and saporin, a type I RIP from soapwort (Saponaria officinalis L.). Two ricin gene sequences were isolated from a Ricinus genomic library and partially characterised. One gene was a badly damaged ricin-like pseudogene whilst the other was shown to encode an active polypeptide. A second ricin sequence encoding an active polypeptide was isolated using Polymerase Chain Reaction (PGR) DNA amplification. The specificity of PGR amplification was investigated using the ricin and related agglutinin gene sequences. Partial amino acid sequence data derived from protein sequencing of saporin-6 was used to synthesise degenerate inosine-containing oligonucleotides. These directed the PGR amplification of part of the saporin coding sequence from genomic DNA. The product was used as a saporin-specific hybridisation probe. Southern analysis of Saponaria genomic DNA indicated that saporin sequences comprised a small multigene family. Three independent saporin containing genomic clones were isolated from a Saponaria genomic library. Two clones were truncated whilst the third contained a complete saporin coding sequence. The saporin and ricin coding sequences were expressed in vitro and shown to inhibit protein synthesis. Aniline cleavage assays of ribosomal RNA extracted from ribosomes exposed to the products of the RIP coding sequences were carried out. These indicated that the polypeptides encoded by the RIP gene sequences had specific RNA N-glycosidase activity

SNi from SN2: a front-face mechanism ‘synthase’ engineered from a retaining hydrolase

SNi or SNi-like mechanisms, in which leaving group departure and nucleophile approach occur on the same ‘front’ face, have been observed previously experimentally and computationally in both the chemical and enzymatic (glycosyltransferase) substitution reactions of α-glycosyl electrophiles. Given the availability of often energetically comparable competing pathways for substitution (SNi vs SN1 vs SN2) the precise modulation of this archetypal reaction type should be feasible. Here, we show that the drastic engineering of a protein that catalyzes substitution, a retaining β-glycosidase (from Sulfolobus solfataricus SSβG), apparently changes the mode of reaction from “SN2” to “SNi”. Destruction of the nucleophilic Glu387 of SSβG-WT through Glu387Tyr mutation (E387Y) created a catalyst (SSβG-E387Y) with lowered but clear transglycosylation substitution activity with activated substrates, altered substrate and reaction preferences and hence useful synthetic (‘synthase’) utility by virtue of its low hydrolytic activity with unactivated substrates. Strikingly, the catalyst still displayed retaining β stereoselectivity, despite lacking a suitable nucleophile; pH-activity profile, mechanism-based inactivators and mutational analyses suggest that SSβG-E387Y operates without either the use of nucleophile or general acid/base residues, consistent with a SNi or SNi-like mechanism. An x-ray structure of SSβG-E387Y and subsequent metadynamics simulation suggest recruitment of substrates aided by a π-sugar interaction with the introduced Tyr387 and reveal a QM/MM free energy landscape for the substitution reaction catalyzed by this unnatural enzyme similar to those of known natural, SNi-like glycosyltransferase (GT) enzymes. Proton flight from the putative hydroxyl nucleophile to the developing p-nitrophenoxide leaving group of the substituted molecule in the reactant complex creates a hydrogen bond that appears to crucially facilitate the mechanism, mimicking the natural mechanism of SNi-GTs. An oxocarbenium ion-pair minimum along the reaction pathway suggests a step-wise SNi-like DN*ANss rather than a concerted SNi DNAN mechanism. This first observation of a front face mechanism in a β-retaining glycosyl transfer enzyme highlights, not only that unusual SNi reaction pathways may be accessed through direct engineering of catalysts with suitable environments, but also suggests that ‘β-SNi’ reactions are also feasible for glycosyl transfer enzymes and the more widespread existence of SNi or SNi-like mechanism in nature

Unique Regulation of the Active site of the Serine Esterase S-Formylglutathione Hydrolase.

S-Formylglutathione hydrolases (SFGHs) are highly conserved thioesterases present in prokaryotes and eukaryotes, and form part of the formaldehyde detoxification pathway, as well as functioning as xenobiotic-hydrolysing carboxyesterases. As defined by their sensitivity to covalent modification, SFGHs behave as cysteine hydrolases, being inactivated by thiol alkylating agents, while being insensitive to inhibition by organophosphates such as paraoxon. As such, the enzyme has been classified as an esterase D in animals, plants and microbes. While SFGHs do contain a conserved cysteine residue that has been implicated in catalysis, sequence analysis also reveals the classic catalytic triad of a serine hydrolase. Using a combination of selective protein modification and X-ray crystallography, AtSFGH from Arabidospsis thaliana has been shown to be a serine hydrolase rather than a cysteine hydrolase. Uniquely, the conserved reactive cysteine (Cys59) previously implicated in catalysis lies in close proximity to the serine hydrolase triad, serving a gate-keeping function in comprehensively regulating access to the active site. Thus, any covalent modification of Cys59 inhibited all hydrolase activities of the enzyme. When isolated from Escherichia coli, a major proportion of recombinant AtSFGH was recovered with the Cys59 forming a mixed disulfide with glutathione. Reversible disulfide formation with glutathione could be demonstrated to regulate hydrolase activity in vitro. The importance of Cys59 in regulating AtSFGH in planta was demonstrated in transient expression assays in Arabidopsis protoplasts. As determined by fluorescence microscopy, the Cys59Ser mutant enzyme was shown to rapidly hydrolyse 4-methylumbelliferyl acetate in paraoxon-treated cells, while the native enzyme was found to be inactive. Our results clarify the classification of AtSFGHs as hydrolases and suggest that the regulatory and conserved cysteine provides an unusual redox-sensitive regulation to an enzyme functioning in both primary and xenobiotic metabolism in prokaryotes and eukaryotes

A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain

A novel protein phosphatase in Arabidopsis thaliana was identified by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion 'pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways

MptpB, a virulence factor from Mycobacterium tuberculosis, exhibits triple-specificity phosphatase activity

Bacterial pathogens have developed sophisticated mechanisms of evading the immune system to survive in infected host cells. Central to the pathogenesis of Mycobacterium tuberculosis is the arrest of phagosome maturation, partly through interference with PtdIns signalling. The protein phosphatase MptpB is an essential secreted virulence factor in M. tuberculosis. A combination of bioinformatics analysis, enzyme kinetics and substrate-specificity characterization revealed that MptpB exhibits both dual-specificity protein phosphatase activity and, importantly, phosphoinositide phosphatase activity. Mutagenesis of conserved residues in the active site signature indicates a cysteine-based mechanism of dephosphorylation and identifies two new catalytic residues, Asp165, essential in catalysis, and Lys164, apparently involved in substrate specificity. Sequence similarities with mammalian lipid phosphatases and a preference for phosphoinositide substrates suggests a potential novel role of MptpB in PtdIns metabolism in the host and reveals new perspectives for the role of this phosphatase in mycobacteria pathogenicity