Stephen Bornemann

Three Isoforms of Isoamylase Contribute Different Catalytic Properties for the Debranching of Potato Glucans

Isoamylases are debranching enzymes that hydrolyze α-1,6 linkages in α-1,4/α-1,6–linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.

Self-poisoning of Mycobacterium tuberculosis by targeting GlgE in an [alpha]-glucan pathway

New chemotherapeutics are urgently required to control the tuberculosis pandemic. We describe a new pathway from trehalose to alpha-glucan in Mycobacterium tuberculosis comprising four enzymatic steps mediated by TreS, Pep2, GlgE (which has been identified as a maltosyltransferase that uses maltose 1-phosphate) and GlgB. Using traditional and chemical reverse genetics, we show that GlgE inactivation causes rapid death of M. tuberculosis in vitro and in mice through a self-poisoning accumulation of maltose 1-phosphate. Poisoning elicits pleiotropic phosphosugar-induced stress responses promoted by a self-amplifying feedback loop where trehalose-forming enzymes are upregulated. Moreover, the pathway from trehalose to alpha-glucan exhibited a synthetic lethal interaction with the glucosyltransferase Rv3032, which is involved in biosynthesis of polymethylated alpha-glucans, because key enzymes in each pathway could not be simultaneously inactivated. The unique combination of maltose 1-phosphate toxicity and gene essentiality within a synthetic lethal pathway validates GlgE as a distinct potential drug target that exploits new synergistic mechanisms to induce death in M. tuberculosis.

Flavoenzymes that catalyse reactions with no net redox change

This review covers unusual flavoenzymes that catalyse reactions with no net redox change. Some of these enzymes utilise the redox properties of flavin directly in catalysis with either two-electron chemistry (N-methylglutamate synthase and 5-hydroxyvaleryl-CoA dehydratase) or free radical chemistry (chorismate synthase, DNA photolyase, (6-4) photolyase and 4-hydroxybutyryl-CoA dehydratase). Whether the flavin has a redox role in some other flavoproteins is not yet clear ((R)-2-hydroxyacyl-CoA dehydratases, isopentenyl diphosphate isomerase and UDPgalactopyranose mutase). The remaining flavoenzymes do not make use of the redox properties of the flavin (acetohydroxyacid synthases and hydroxynitrile lyase). The literature is reviewed up to early 2002 and 121 references are cited.

Bacillus subtilis YvrK Is an Acid-Induced Oxalate Decarboxylase

Bacillus subtilis has been shown to express a cytosolic oxalate decarboxylase (EC 4.1.1.2). The enzyme was induced in acidic growth media, particularly at pH 5.0, but not by oxalate. The enzyme was purified, and N-terminal sequencing identified the protein to be encoded by yvrK. The role of the first oxalate decarboxylase to be identified in a prokaryote is discussed.

Unexpected and widespread connections between bacterial glycogen and trehalose metabolism

Glycogen, a large α-glucan, is a ubiquitous energy storage molecule among bacteria, and its biosynthesis by the classical GlgC-GlgA pathway and its degradation have long been well understood - or so we thought. A second pathway of α-glucan synthesis, the four-step GlgE pathway, was recently discovered in mycobacteria. It requires trehalose as a precursor, and has been genetically validated as a novel anti-tuberculosis drug target. The ability to convert glycogen into trehalose was already known, so the GlgE pathway provides a complementary way of cycling these two metabolites. As well as containing cytosolic storage glycogen, mycobacteria possess an outer capsule containing a glycogen-like α-glucan that is implicated in immune system evasion, so the GlgE pathway might be linked to capsular α-glucan biosynthesis. Another pathway (the Rv3032 pathway) for α-glucan biosynthesis in mycobacteria generates a methylglucose lipopolysaccharide thought to be associated with fatty acid metabolism. A comparative genomic analysis was carried out to evaluate the occurrence and role of the classical pathway, the new GlgE pathway and the Rv3032 pathway across bacteria occupying very different ecological niches. The GlgE pathway is represented in 14 % of sequenced genomes from diverse bacteria (about half as common as the classical pathway), while the Rv3032 pathway is restricted with few exceptions to mycobacteria, and the GlgB branching enzyme, usually presumed to be associated with the classical pathway, correlates more strongly with the new GlgE pathway. The microbiological implications of recent discoveries in the light of the comparative genomic analysis are discussed.

Calcium/Calmodulin-Dependent Protein Kinase Is Negatively and Positively Regulated by Calcium, Providing a Mechanism for Decoding Calcium Responses during Symbiosis Signaling

This study dissects the regulation of a calcium/calmodulin-dependent protein kinase (CCaMK) during symbiotic signaling and reveals that CCaMK is both negatively and positively regulated by calcium to create a robust molecular switch that is responsive to calcium concentrations associated with both the basal state and with oscillations. The establishment of symbiotic associations in plants requires calcium oscillations that must be decoded to invoke downstream developmental programs. In animal systems, comparable calcium oscillations are decoded by calmodulin (CaM)–dependent protein kinases, but symbiotic signaling involves a calcium/CaM–dependent protein kinase (CCaMK) that is unique to plants. CCaMK differs from the animal CaM kinases by its dual ability to bind free calcium, via calcium binding EF-hand domains on the protein, or to bind calcium complexed with CaM, via a CaM binding domain. In this study, we dissect this dual regulation of CCaMK by calcium. We find that calcium binding to the EF-hand domains promotes autophosphorylation, which negatively regulates CCaMK by stabilizing the inactive state of the protein. By contrast, calcium-dependent CaM binding overrides the effects of autophosphorylation and activates the protein. The differential calcium binding affinities of the EF-hand domains compared with those of CaM suggest that CCaMK is maintained in the inactive state at basal calcium concentrations and is activated via CaM binding during calcium oscillations. This work provides a model for decoding calcium oscillations that uses differential calcium binding affinities to create a robust molecular switch that is responsive to calcium concentrations associated with both the basal state and with oscillations.

Bacillus subtilis YxaG is a novel Fe-containing quercetin 2,3-dioxygenase

The Bacillus subtilis genome contains genes for three hypothetical proteins belonging to the bicupin family, two of which we have previously shown to be Mn(II)‐dependent oxalate decarboxylases. We have now shown that the third, YxaG, exhibits quercetin 2,3‐dioxygenase activity and that it contains Fe ions. This contrasts with the eukaryotic enzyme which contains a Cu ion. YxaG is the first prokaryotic carbon monoxide‐forming enzyme that utilises a flavonol to be characterised and is only the second example of a prokaryotic dioxygenolytic carbon monoxide‐forming enzyme known to contain a cofactor. It is proposed to rename the B. subtilis gene qdoI.

Structure of Streptomyces Maltosyltransferase GlgE, a Homologue of a Genetically Validated Anti-tuberculosis Target

Background: GlgE is a maltosyltransferase involved in bacterial α-glucan biosynthesis and is a genetically validated anti-tuberculosis target. Results: We have determined the catalytic properties of Streptomyces coelicolor GlgE and solved its structure. Conclusion: The enzyme has the same catalytic properties as Mycobacterium tuberculosis GlgE and the structure reveals how GlgE functions. Significance: The structure will help guide the development of inhibitors with therapeutic potential. GlgE is a recently identified (1→4)-α-d-glucan:phosphate α-d-maltosyltransferase involved in α-glucan biosynthesis in bacteria and is a genetically validated anti-tuberculosis drug target. It is a member of the GH13_3 CAZy subfamily for which no structures were previously known. We have solved the structure of GlgE isoform I from Streptomyces coelicolor and shown that this enzyme has the same catalytic and very similar kinetic properties to GlgE from Mycobacterium tuberculosis. The S. coelicolor enzyme forms a homodimer with each subunit comprising five domains, including a core catalytic α-amylase-type domain A with a (β/α)8 fold. This domain is elaborated with domain B and two inserts that are specifically configured to define a well conserved donor pocket capable of binding maltose. Domain A, together with domain N from the neighboring subunit, forms a hydrophobic patch that is close to the maltose-binding site and capable of binding cyclodextrins. Cyclodextrins competitively inhibit the binding of maltooligosaccharides to the S. coelicolor enzyme, showing that the hydrophobic patch overlaps with the acceptor binding site. This patch is incompletely conserved in the M. tuberculosis enzyme such that cyclodextrins do not inhibit this enzyme, despite acceptor length specificity being conserved. The crystal structure reveals two further domains, C and S, the latter being a helix bundle not previously reported in GH13 members. The structure provides a framework for understanding how GlgE functions and will help guide the development of inhibitors with therapeutic potential.

The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations.

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Delta162-163 or Delta162-164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Delta162-163 and S161A) strongly support the hypothesis that site 2 is purely structural.

pH-Dependent Structures of the Manganese Binding Sites in Oxalate Decarboxylase as Revealed by High-Field Electron Paramagnetic Resonance

A high-field electron paramagnetic resonance (HFEPR) study of oxalate decarboxylase (OxdC) is reported. OxdC breaks down oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site-1 and -2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site -2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described by a single pK(a). This pH dependence was independent of the presence of the His-tag and of whether the preparations contained 1.2 or 1.6 Mn per subunit. Possible structures of the six species are proposed based on spectroscopic data from model complexes and existing protein crystallographic structures obtained at pH 8 are discussed. Although site-1 has been identified as the active site and no role has been assigned to site-2, the pronounced changes in the electronic structure of the latter and its pH behavior, which also matches the pH-dependent activity of this enzyme, suggests that even if the conversion of oxalate to formate is carried out at site-1, site-2 likely plays a catalytically relevant role.