Shirley A. Fairhurst

Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate

An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate‐lyase enzyme Pfl revealed the existence of a weak, cross‐reacting 85 kDa polypeptide that exhibited the characteristic oxygen‐dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross‐reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L‐serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate‐lyase and 2‐ketobutyrate formate‐lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L‐threonine to propionate. These studies confirmed that 2‐ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl‐CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate‐dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl‐CoA and propionyl‐phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl‐P to propionate by AckA or TdcD.

Pseudomonas sax Genes Overcome Aliphatic Isothiocyanate–Mediated Non-Host Resistance in Arabidopsis

Natural-product effectors of disease resistance in Arabidopsis reveal complementary disabling mechanisms in the pathogen. Most plant-microbe interactions do not result in disease; natural products restrict non-host pathogens. We found that sulforaphane (4-methylsulfinylbutyl isothiocyanate), a natural product derived from aliphatic glucosinolates, inhibits growth in Arabidopsis of non-host Pseudomonas bacteria in planta. Multiple sax genes (saxCAB/F/D/G) were identified in Pseudomonas species virulent on Arabidopsis. These sax genes are required to overwhelm isothiocyanate-based defenses and facilitate a disease outcome, especially in the young leaves critical for plant survival. Introduction of saxCAB genes into non-host strains enabled them to overcome these Arabidopsis defenses. Our study shows that aliphatic isothiocyanates, previously shown to limit damage by herbivores, are also crucial, robust, and developmentally regulated defenses that underpin non-host resistance in the Arabidopsis-Pseudomonas pathosystem.

The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine.

The Elp3 subunit of the Elongator complex is highly conserved from archaea to humans and contains a well‐characterized C‐terminal histone acetyltransferase (HAT) domain. The central region of Elp3 shares significant sequence homology to the Radical SAM superfamily. Members of this large family of bacterial proteins contain a FeS cluster and use S‐adenosylmethionine (SAM) to catalyse a variety of radical reactions. To biochemically characterize this domain we have expressed and purified the corresponding fragment of the Methanocaldococcus jannaschii Elp3 protein. The presence of a Fe4S4 cluster has been confirmed by UV‐visible spectroscopy and electron paramagnetic resonance (EPR) spectroscopy and the Fe content determined by both a colorimetric assay and atomic absorption spectroscopy. The cysteine residues involved in cluster formation have been identified by site‐directed mutagenesis. The protein binds SAM and the binding alters the EPR spectrum of the FeS cluster. Our results provide biochemical support to the hypothesis that Elp3 does indeed contain the Fe4S4 cluster which characterizes the Radical SAM superfamily and binds SAM, suggesting that Elp3, in addition to its HAT activity, has a second as yet uncharacterized catalytic function. We also present preliminary data to show that the protein cleaves SAM.

All-iron hydrogenase: synthesis, structure and properties of {2Fe3S}-assemblies related to the di-iron sub-site of the H-clusterElectronic supplementary information (ESI) available: crystal and structure refinement data for complexes 4a, 4b and 5a. See http://www.rsc.org/suppdata/dt/b2/b209690k/

Tripodal dithiolate thioether ligands MeC(CH2SH)2CH2SR (R = Me or Ph) provide a route to {2Fe3S}-complexes and syntheses are described. X-Ray crystal structures for two {2Fe3S}-pentacarbonyl derivatives and that for the first carbonyl cyanide are reported, together with temperature dependent 1H-NMR, Mossbauer, FTIR and redox potential data. The NMR data establish fluctionality associated with inversion at the thioether sulfur in the carbonyl complexes. The Mossbauer data affirm that the coordination environment of the two iron atoms in a dicyanide bridging carbonyl intermediate are differentiated. Bridging carbonyl intermediates have been structurally and spectroscopically identified in resting and CO inhibited forms of the sub-site of all-iron hydrogenases; the observation of a thermally unstable {2Fe3S}-bridging carbonyl intermediate is discussed in this context.

A Novel Polyamine Acyltransferase Responsible for the Accumulation of Spermidine Conjugates in Arabidopsis Seed

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylspermidine and N,N′-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

Significance We carried out functional analysis of the oat enzyme AsCYP51H10, which is a divergent member of the CYP51 cytochrome P450 family and showed that this enzyme is able to catalyze both hydroxylation and epoxidation of the simple triterpene β-amyrin to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). In contrast, the canonical CYP51 enzymes are highly conserved and catalyze only sterol demethylation. We further show that the C12,13 epoxy group is critical for antifungal activity, a discovery that has important implications for triterpene metabolic engineering for food, health, and industrial biotechnology applications. Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family—as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.

Thiazole synthase from Escherichia Coli: An investigation of the substrates and purified proteins required for activity In Vitro

Thiamine is biosynthesized by combining two heterocyclic precursors. In Escherichia coli and other anaerobes, one of the heterocycles, 4-methyl-5-(β-hydroxyethyl) thiazole phosphate, is biosynthesized from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine. Genetic evidence has identified thiH, thiG, thiS, and thiF as essential for thiazole biosynthesis in E. coli. In this paper, we describe the measurement of the thiazole phosphate-forming reaction using purified protein components. The activity is shown to require four proteins isolated as heterodimers: ThiGH and ThiFS. Reconstitution of the [4Fe-4S] cluster in ThiH was essential for activity, as was the use of ThiS in the thiocarboxylate form. Spectroscopic studies with ThiGH strongly suggested that S-adenosylmethionine (AdoMet) bound to the [4Fe-4S] cluster, which became more susceptible to reduction to the +1 state. Assays of thiazole phosphate formation showed that, in addition to the proteins, Dxp, tyrosine, AdoMet, and a reductant were required. The analysis showed that no more than 1 mol eq of thiazole phosphate was formed per ThiGH. Furthermore, for each mole of thiazole-P formed, 1 eq of AdoMet and 1 eq of tyrosine were utilized, and 1 eq of 5′-deoxyadenosine was produced. These results demonstrate that ThiH is a member of the “radical-AdoMet” family and support a mechanistic hypothesis in which AdoMet is reductively cleaved to yield a highly reactive 5′-deoxyadenosyl radical. This radical is proposed to abstract the phenolic hydrogen atom from tyrosine, and the resultant substrate radical cleaves to yield dehydroglycine, which is required by ThiG for the thiazole cyclization reaction.

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.

Electron spin resonance study of CH3CNSSN˙, C6H5CNSSN˙, and SNSSN˙+ free radicals

Isotropic and powder e.s.r. spectra have been recorded for CH3[graphic omitted]˙, C6H5[graphic omitted]˙, and [graphic omitted]˙+. Isotropic labelling with nitrogen-15 and sulphur-33 has been accomplished for [graphic omitted]˙+ and it has been possible to prepare 33[graphic omitted]˙+. Sulphur-33 satellites have been observed for C6H5[graphic omitted]˙. MNDO and Gaussian 76 calculations have been used to calculate the minimum-energy structures of the radicals, while INDO calculations have provided values for the hyperfine coupling constants. Unfortunately, poor agreement was obtained between the latter and the corresponding experimental values. All the radicals dimerise in solution at low temperatures and we have been able to measure the energetics of dimerisation for C6H5[graphic omitted]˙ and [graphic omitted]˙+. The dimers exist as crystalline solids which contain readily detectable amounts of the monomeric free radical.

Thermally induced structural changes in glycinin, the 11S globulin of soya bean (Glycine max)—an in situ spectroscopic study

The thermal denaturation behaviour of glycinin solutions has been studied in situ as a function of ionic strength using various spectroscopic methods. Changes in secondary structure occurred at temperatures above 60 degrees C, well before the onset of gelation. Even after heating to 95 degrees C, much of the native beta-sheet structure of glycinin was retained, as indicated by the amide I peak maximum at 1635 cm(-1) in the Fourier transformed infrared (FT-IR) spectrum. This was accompanied by an increase in the 1625 cm(-1) band, indicative of the formation of intermolecular beta-sheet associated with protein aggregation. Nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of highly mobile regions in glycinin comprising predominantly of Gln and Glu residues, corresponding to mobile regions previously identified by crystallographic studies. There was also evidence of a hydrogen-bonded structure within this mobile region, which may correspond to an alpha-helical region from Pro(256) to (or just before) Pro(269) in proglycinin. This structure disappeared at 95 degrees C, when heat-set gel formation occurred, as indicated by a sudden broadening and weakening of the NMR signal. Otherwise the NMR spectrum changed little during heating, emphasising the remarkable thermal stability of glycinin. It is proposed that during heating the core beta-barrel structure remains intact, but that the interface between the beta-domains melts, revealing hydrophobic faces which may then form new structures in a gel-network. As Cys(45), which forms the disulfide with Cys(12) linking the acidic and basic polypeptides, is found in this interface, such a rearrangement of the individual beta-domains could be accompanied by cleavage of this disulfide bond, as is observed experimentally. Such information contributes to our understanding the aggregative behaviour of proteins, and hence develops knowledge-based strategies for controlling and manipulating it.