Oleate Hydratase Catalyzes the Hydration of a Nonactivated Carbon-Carbon Bond

The hydration of oleic acid into 10-hydroxystearic acid was originally described for a Pseudomonas cell extract almost half a century ago. In the intervening years, the enzyme has never been characterized in any detail. We report here the isolation and characterization of oleate hydratase (EC 4.2.1.53) from Elizabethkingia meningoseptica.The ability of cells to convert oleic acid (OA) into 10-hydroxystearic acid (10-HSA) was discovered by Wallen et al. in Pseudomonas sp. strain 3266 in 1962 (Fig. ​(Fig.1)1) (12). In the following years, many other strains were identified that were also able to convert OA into 10-HSA or to further oxidize it to 10-ketostearic acid (2, 3, 5, 7). The Pseudomonas cells generally start to produce optically pure d-10-HSA in the stationary growth phase, and they do not seem to metabolize it any further, since levels of product accumulate in the fermentation broth. The putative enzyme for this conversion is referred to as oleate hydratase (EC 4.2.1.53); however, so far it has not been purified or characterized in any detail.Open in a separate windowFIG. 1.Reaction catalyzed by oleate hydratase; the conversion of OA into 10-HSA.Kinetic studies have been performed with cell extracts, giving some insight into the stereospecificity and the possible mechanism of the reaction. Studies with ¹⁸O-labeled water reported the incorporation of ¹⁸O at the C-10 position of 10-HSA, confirming a hydration mechanism (7). The reaction was shown to be reversible; however, the detected concentration ratio at equilibrium was always in the range of 85:15 in favor of 10-HSA (9).Here we report the isolation and first biochemical characterization of the oleate hydratase protein from Elizabethkingia meningoseptica (formerly known as Pseudomonas sp. strain 3266).The primer set GM3 and GM4 (8) was used for PCR amplification of the Pseudomonas sp. strain 3266 16S-rRNA genes. The product (1,444 bp) was sequenced, and 16S phylogeny analysis resulted in a unanimous determination of the species as Elizabethkingia (Chryseobacterium) meningoseptica with a >99.8% resemblance.     

耐底物腈水合酶融合子的产酶条件优化

采用正交设计法对耐底物腈水合酶融合子的发酵条件进行优化,以发酵液起始pH,发酵周期,接种量,装料系数作为考察因素,最终确定最佳发酵条件为:起始pH8.0、发酵周期54h、接种量12％、装液系数12％.在此优化条件下融合子腈水合酶的活力达到1100万U/ml,较优化前提高了83.3％.通过响应面法对发酵培养基配方进行优化研究,采用Plackett-Burman法对8个因素进行了筛选,结果表明,葡萄糖、尿素、磷酸氢二钾、磷酸二氢钾是影响发酵液腈水合酶产量的主效应因子.用最陡爬坡试验及Central composite design设计进一步优化,利用Design-Expert软件进行二次回归分析,得到各因素的最佳浓度为:葡萄糖22.62g/L、尿素9.76g/L、K2HP04 1.22g/L、KH2PO41.268g/L.在此培养基优化配方下融合子腈水合酶的活力达到1280万U/ml,较原配方的酶活提高了16.4％.     

HrpW of Erwinia amylovora,a New Harpin That Contains a Domain Homologous to Pectate Lyases of a Distinct Class

Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream of hrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwinia species, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts.Most gram-negative plant-pathogenic bacteria contain clusters of genes termed hrp that are required for elicitation of a rapid localized defense response called the hypersensitive reaction (HR) in incompatible plants and that are required for pathogenicity in susceptible plants (1). Proteins encoded by hrp genes are involved in the regulation of the expression of other hrp genes and in a specialized secretion process called the Hrp or type III pathway (9). Harpins, a major class of proteins that travel the pathway (including HrpN of Erwinia species, HrpZ of Pseudomonas syringae, and PopA of Ralstonia solanacearum), elicit the HR when infiltrated into the apoplast of leaf tissue (reference 1 and references therein). They are heat stable, rich in Gly and/or Ser, lack Cys, and differ in their primary sequences. In Erwinia amylovora, mutation of hrpN results in substantially reduced Hrp phenotype (4, 6, 45).E. amylovora causes the devastating fire-blight disease on many rosaceous plants, such as apple, pear, and cotoneaster. Cosmids pCPP430 and pCPP450, which harbor the hrp gene cluster of E. amylovora Ea321, enable Escherichia coli to elicit the HR in tobacco (7). The region of pCPP430 essential for the Hrp phenotype encodes two-component regulatory proteins, a ς⁵⁴ enhancer-binding protein, a sigma factor, secretory proteins, and the HrpN harpin (11, 27, 42–45). In contrast, the locus next to hrp genes, designated dsp, contains pathogenicity genes, and P. syringae pv. glycinea containing the E. amylovora dsp locus causes the HR rather than disease in soybean plants (10). This locus encodes a Hrp-secreted protein and a probable chaperone of the secreted protein (8, 10, 17).Additional HR elicitors in E. amylovora have been suspected based on the HR-variable phenotype of E. amylovora hrpN mutants (references 4 and 6; see also Table ​Table1).1). We report here the identification and characterization of a novel harpin of E. amylovora, HrpW, the C-terminal domain of which surprisingly is homologous to fungal pectate lyases (PLs). We show that HrpW, the production of which is controlled by hrpL, is delivered by the E. amylovora Hrp pathway. HrpW elicits the HR in plants, and the HR necrosis is not due to the potential PL activity of HrpW. Finally, we provide evidence that HrpW is not required for the HR and pathogenicity, although when overexpressed it enhances the HR-eliciting activity of a hrpN mutant. Preliminary reports on E. amylovora HrpW have been made (28, 29), and, while this article was under revision, a paper describing HrpW from E. amylovora CFBP1430 (16) appeared.

TABLE 1

HR induction and virulence of E. amylovora Ea321 and mutant derivatives^a

Molybdoenzyme That Catalyzes the Anaerobic Hydroxylation of a Tertiary Carbon Atom in the Side Chain of Cholesterol

Cholesterol is a ubiquitous hydrocarbon compound that can serve as substrate for microbial growth. This steroid and related cyclic compounds are recalcitrant due to their low solubility in water, complex ring structure, the presence of quaternary carbon atoms, and the low number of functional groups. Aerobic metabolism therefore makes use of reactive molecular oxygen as co-substrate of oxygenases to hydroxylate and cleave the sterane ring system. Consequently, anaerobic metabolism must substitute oxygenase-catalyzed steps by O₂-independent hydroxylases. Here we show that one of the initial reactions of anaerobic cholesterol metabolism in the β-proteobacterium Sterolibacterium denitrificans is catalyzed by an unprecedented enzyme that hydroxylates the tertiary C25 atom of the side chain without molecular oxygen forming a tertiary alcohol. This steroid C25 dehydrogenase belongs to the dimethyl sulfoxide dehydrogenase molybdoenzyme family, the closest relative being ethylbenzene dehydrogenase. It is a heterotrimer, which is probably located at the periplasmic side of the membrane and contains one molybdenum cofactor, five [Fe-S] clusters, and one heme b. The draft genome of the organism contains several genes coding for related enzymes that probably replace oxygenases in steroid metabolism.     

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (P_i) residues linked by “high-energy” phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities.     

Mutations That Improve the Binding of Yeast Flp Recombinase to Its Substrate

When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.     

New Field Isolates of Rhizobium leguminosarum Biovar Viciae That Nodulate the Primitive Pea Cultivar Afghanistan in Addition to Modern Cultivars

A collection of 13 field isolates of Rhizobium leguminosarum bv. viciae that have the ability to nodulate the roots of current North American cultivars of peas as well as a “primitive” cultivar, Afghanistan, was examined. These isolates originated in diverse geographical regions of the world, which indicates that this phenotype is not restricted to isolates from any one region. When subclones of the nodulation region from one plasmid were used to examine EcoRI-fragment-length polymorphisms in this collection of strains as well as in a collection comprising strains that do not nodulate the primitive cultivar, polymorphism was found in both collections. With one exception, RisA6, all strains that nodulated cv. Afghanistan pea contained a region called nodX as an extension to the nodA BCIJ operon that has been observed in all R. leguminosarum bv. viciae strains, including those that do not nodulate cv. Afghanistan pea. RisA6 was also the only strain in which nodulating ability could not be associated with a conjugative plasmid.     

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.     

Cloning and Mapping of the cDNA for Human Sarcosine Dehydrogenase, a Flavoenzyme Defective in Patients with Sarcosinemia

Sarcosine dehydrogenase is a liver mitochondrial matrix flavoenzyme that is defective in patients with sarcosinemia, a rare autosomal metabolic defect characterized by elevated levels of sarcosine in blood and urine. Some patients also exhibit mental retardation and growth failure. A full-length cDNA for human sarcosine dehydrogenase was isolated from an adult liver cDNA library. The first 22 residues in the deduced amino acid sequence exhibit features expected for a mitochondrial targeting sequence. The predicted mass of the mature human liver sarcosine dehydrogenase (99,505 Da) is in good agreement with that observed for rat liver sarcosine dehydrogenase ( approximately 100,000 Da). Human sarcosine dehydrogenase exhibits 89% identity with rat liver sarcosine dehydrogenase and strong homology ( approximately 35% identity) with rat liver dimethylglycine dehydrogenase, a sarcosine dehydrogenase-related protein from Rhodobacter capsulatus, and the regulatory subunit from bovine pyruvate dehydrogenase phosphatase. The human sarcosine dehydrogenase gene is at least 75.3 kb long and located on chromosome 9q34. The adult human liver clone is assembled from 21 exons (1-6, 7a, 8a, 9-21). Two smaller cDNA clones, isolated from adult liver and infant brain libraries, were assembled from the same sarcosine dehydrogenase gene by the use of alternate polyadenylation and splice sites. This is the first report of the genomic structure of the sarcosine dehydrogenase gene in any species. The observed chromosomal location is consistent with genetic studies with a mouse model for sarcosinemia that map the mouse gene to a region of mouse chromosome 2 syntenic with human 9q33-q34. The availability of the SDH gene sequence will enable characterization of the genotypes of sarcosinemia patients with different phenotypes.     

2-Haloacrylate reductase, a novel enzyme of the medium chain dehydrogenase/reductase superfamily that catalyzes the reduction of a carbon-carbon double bond of unsaturated organohalogen compounds

A soil bacterium, Burkholderia sp. WS, grows on 2-chloroacrylate as the sole carbon source. To identify the enzymes metabolizing 2-chloroacrylate, we carried out comparative two-dimensional gel electrophoresis of the proteins from 2-chloroacrylate- and lactate-grown bacterial cells. As a result, we found that a protein named CAA43 was inducibly synthesized when the cells were grown on 2-chloroacrylate. The CAA43 gene was cloned and shown to encode a protein of 333 amino acid residues (M(r) 35,788) that shared a significant sequence similarity with NADPH-dependent quinone oxidoreductase from Escherichia coli (38.2% identity). CAA43 was overproduced in E. coli and purified to homogeneity. The purified protein catalyzed the NADPH-dependent reduction of the carbon-carbon double bond of 2-chloroacrylate to produce (S)-2-chloropropionate, which is probably further metabolized to (R)-lactate by (S)-2-haloacid dehalogenase in Burkholderia sp. WS. NADH did not serve as a reductant. Despite the sequence similarity to quinone oxidoreductases, CAA43 did not act on 1,4-benzoquinone and 1,4-naphthoquinone. 2-Chloroacrylate analogs, such as acrylate and methacrylate, were also inert as the substrates. In contrast, 2-bromoacrylate served as the substrate. Thus, we named this novel enzyme 2-haloacrylate reductase. This study revealed a new pathway for the degradation of unsaturated organohalogen compounds. It is also notable that the enzyme is useful for the production of (S)-2-chloropropionate, which is used for the industrial production of aryloxyphenoxypropionic acid herbicides.     

Xerasia meschniggi (Reitter, 1905) (Coleoptera,Byturidae), a New Addition to the Russian Fauna

Entomological Review - Xerasia meschniggi (Reitter, 1905) (Byturidae), previously considered endemic to the south of Central Europe, was discovered among material of the beetles collected by the...     

1+1?=?3: A Fusion of 2 Enzymes in the Methionine Salvage Pathway of Tetrahymena thermophila Creates a Trifunctional Enzyme That Catalyzes 3 Steps in the Pathway

The methionine salvage pathway is responsible for regenerating methionine from its derivative, methylthioadenosine. The complete set of enzymes of the methionine pathway has been previously described in bacteria. Despite its importance, the pathway has only been fully described in one eukaryotic organism, yeast. Here we use a computational approach to identify the enzymes of the methionine salvage pathway in another eukaryote, Tetrahymena thermophila. In this organism, the pathway has two fused genes, MTNAK and MTNBD. Each of these fusions involves two different genes whose products catalyze two different single steps of the pathway in other organisms. One of the fusion proteins, mtnBD, is formed by enzymes that catalyze non-consecutive steps in the pathway, mtnB and mtnD. Interestingly the gene that codes for the intervening enzyme in the pathway, mtnC, is missing from the genome of Tetrahymena. We used complementation tests in yeast to show that the fusion of mtnB and mtnD from Tetrahymena is able to do in one step what yeast does in three, since it can rescue yeast knockouts of mtnB, mtnC, or mtnD. Fusion genes have proved to be very useful in aiding phylogenetic reconstructions and in the functional characterization of genes. Our results highlight another characteristic of fusion proteins, namely that these proteins can serve as biochemical shortcuts, allowing organisms to completely bypass steps in biochemical pathways.     

Characterization of a New Bacillus thuringiensis Strain That Is Toxic to Coleoptera

A new strain of Bacillus thuringiensis 2-7 was found to belong to the serotype H8. Cells of this strain contained irregular and flat crystalline inclusions and two large plasmids. The gene responsible for crystal formation is most likely located on the large plasmid greater than 105 MDa in size. Comparison of the cry gene of B. thuringiensis 2-7 and the cryIIIA gene of B. thuringiensis subsp. tenebrionis showed that their nucleotide sequences are identical.     

Phenotypic and Genetic Analysis of det2, a New Mutant That Affects Light-Regulated Seedling Development in Arabidopsis

The greening phenotypes produced by recessive mutations in a gene designated de-etiolated-2 (DET2) are described. Recessive mutations in the DET2 gene uncouple light signals from a number of light-dependent processes. det2 mutations result in dark-grown Arabidopsis thaliana seedlings with many characteristics of light-grown plants, including hypocotyl growth inhibition, cotyledon expansion, primary leaf initiation, anthocyanin accumulation, and derepression of light-regulated gene expression. In contrast to these morphological and gene expression changes, however, the chloroplast development program is not initiated in the dark in det2 mutants, suggesting that light-regulated gene expression precedes the differentiation of etioplasts to chloroplasts. det2 mutations thus reveal at least two classes of downstream light-regulated responses that differ in their timing and control mechanisms. Homozygous det2 mutations also affect photoperiodic responses in light-grown plants, including timing of flowering, dark adaptation of gene expression, and onset of leaf senescence. The phenotype of det1 det2 double mutants is additive, implying that DET1 and DET2 function in distinct pathways that affect downstream light-regulated genes. Furthermore, these pathways are not utilized solely during early seedling development but must also be required to regulate different aspects of the light developmental program during later stages of vegetative growth.     

New Atropisomeric Amino Alcohol Ligands for Enantioselective Addition of Diethylzinc to Aldehydes

Efficient synthesis of several new atropisomeric amino alcohols having 1‐phenyl‐1H‐pyrrole skeleton are reported. Steric arrangements of the products were confirmed by a single‐crystal X‐ray measurement. The consequences of the size of the N‐substituents on enantioinduction were examined by employing the enantioselective catalytic addition of diethylzinc to a series of substituted benzaldehydes (yields 91–97%, up to 85% enantiomeric excess). The special effect of the ortho methoxy group of the substrate on the enantioinduction is also interpreted. Chirality 27:216–222, 2015. © 2014 Wiley Periodicals, Inc.