Biochemical properties and in planta effects of NopM, a rhizobial E3 ubiquitin ligase.

Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro Bimolecular fluorescence complementation analysis provided clues about NopM-NopM interactions at plasma membranes in planta NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid-induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.

Characterization of cis-4-hydroxy-D-proline dehydrogenase from Sinorhizobium meliloti.

The hypO gene from Sinorhizobium meliloti, located within the trans-4-hydroxy-L-proline metabolic gene cluster, was first successfully expressed in the host Pseudomonas putida. Purified HypO protein functioned as a FAD-containing cis-4-hydroxy-D-proline dehydrogenase with a homomeric structure. In contrast to other known enzymes, significant activity for D-proline was found, confirming a previously proposed potential involvement in D-proline metabolism.

Dissection of membrane-binding and -remodeling regions in two classes of bacterial phospholipid N-methyltransferases.

Bacterial phospholipid N-methyltransferases (Pmts) catalyze the formation of phosphatidylcholine (PC) via successive N-methylation of phosphatidylethanolamine (PE). They are classified into Sinorhizobium-type and Rhodobacter-type enzymes. The Sinorhizobium-type PmtA protein from the plant pathogen Agrobacterium tumefaciens is recruited to anionic lipids in the cytoplasmic membrane via two amphipathic helices called αA and αF. Besides its enzymatic activity, PmtA is able to remodel membranes mediated by the αA domain. According to the Heliquest program, αA- and αF-like amphipathic helices are also present in other Sinorhizobium- and Rhodobacter-type Pmt enzymes suggesting a conserved architecture of α-helical membrane-binding regions in these methyltransferases. As representatives of the two Pmt families, we investigated the membrane binding and remodeling capacity of Bradyrhizobium japonicum PmtA (Sinorhizobium-type) and PmtX1 (Rhodobacter-type), which act cooperatively to produce PC in consecutive methylation steps. We found that the αA regions in both enzymes bind anionic lipids similar to αA of A. tumefaciens PmtA. Membrane binding of PmtX1 αA is enhanced by its substrate monomethyl-PE indicating a substrate-controlled membrane association. The αA regions of all investigated enzymes remodel spherical liposomes into tubular filaments suggesting a conserved membrane-remodeling capacity of bacterial Pmts. Based on these results we propose that the molecular details of membrane-binding and remodeling are conserved among bacterial Pmts.

The type 3 effector NopL of Sinorhizobium sp. strain NGR234 is a mitogen-activated protein kinase substrate.

Pathogenic bacteria utilize type 3 secretion systems to inject type 3 effectors (T3Es) into host cells, thereby subverting host defense reactions. Similarly, T3Es of symbiotic nitrogen-fixing rhizobia can affect nodule formation on roots of legumes. Previous work showed that NopL (nodulation outer protein L) of Sinorhizobium(Ensifer) sp. strain NGR234 is multiply phosphorylated in eukaryotic cells and that this T3E suppresses responses mediated by mitogen-activated protein (MAP) kinase signaling in yeast (mating pheromone signaling) and plant cells (expression of pathogenesis-related defense proteins). Here, we show that NopL is a MAP kinase substrate. Microscopic observations of fluorescent fusion proteins and bimolecular fluorescence complementation analysis in onion cells indicated that NopL is targeted to the nucleus and forms a complex with SIPK (salicylic acid-induced protein kinase), a MAP kinase of tobacco. In vitro experiments demonstrated that NopL is phosphorylatyed by SIPK. At least nine distinct spots were observed after two-dimensional gel electrophoresis, indicating that NopL can be hyperphosphorylated by MAP kinases. Senescence symptoms in nodules of beans (Phaseolus vulgaris cv. Tendergreen) were analyzed to determine the symbiotic effector activity of different NopL variants with serine to alanine substitutions at identified and predicted phosphorylation sites (serine-proline motif). NopL variants with six or eight serine to alanine substitutions were partially active, whereas NopL forms with 10 or 12 substituted serine residues were inactive. In conclusion, our findings provide evidence that NopL interacts with MAP kinases and reveals the importance of serine-proline motifs for effector activity during symbiosis.

Possible Role of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Activity of Sinorhizobium sp. BL3 on Symbiosis with Mung Bean and Determinate Nodule Senescence.

Sinorhizobium sp. BL3 forms symbiotic interactions with mung bean (Vigna radiata) and contains lrpL-acdS genes, which encode the 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme that cleaves ACC, a precursor of plant ethylene synthesis. Since ethylene interferes with nodule formation in some legumes and plays a role in senescence in plant cells, BL3-enhancing ACC deaminase activity (BL3(+)) and defective mutant (BL3(-)) strains were constructed in order to investigate the effects of this enzyme on symbiosis and nodule senescence. Nodulation competitiveness was weaker in BL3(-) than in the wild-type, but was stronger in BL3(+). The inoculation of BL3(-) into mung bean resulted in less plant growth, a lower nodule dry weight, and smaller nodule number than those in the wild-type, whereas the inoculation of BL3(+) had no marked effects. However, similar nitrogenase activity was observed with all treatments; it was strongly detected 3 weeks after the inoculation and gradually declined with time, indicating senescence. The rate of plant nodulation by BL3(+) increased in a time-dependent manner. Nodules occupied by BL3(-) formed smaller symbiosomes, and bacteroid degradation was more prominent than that in the wild-type 7 weeks after the inoculation. Changes in biochemical molecules during nodulation were tracked by Fourier Transform Infrared (FT-IR) microspectroscopy, and the results obtained confirmed that aging processes differed in nodules occupied by BL3 and BL3(-). This is the first study to show the possible role of ACC deaminase activity in senescence in determinate nodules. Our results suggest that an increase in ACC deaminase activity in this strain does not extend the lifespan of nodules, whereas the lack of this activity may accelerate nodule senescence.

Reactivity of sorbose dehydrogenase from Sinorhizobium sp. 97507 for 1,5-anhydro-D-glucitol.

Purified recombinant sorbose dehydrogenase from Sinorhizobium sp. 97507 exhibited high reactivity for 1,5-anhydro-D-glucitol (1,5-AG) and L-sorbose, but little activity for the other sugars or sugar alcohols tested. Kinetic analysis revealed that its catalytic efficiency (k(cat)/Km) for L-sorbose and 1,5-AG is 1.8 × 10(2) and 1.5 × 10(2) s(-1)·M(-1), respectively.

A Rhizobium selenitireducens protein showing selenite reductase activity.

Biobarriers remove, via precipitation, the metalloid selenite (SeO3⁻²) from groundwater; a process that involves the biological reduction of soluble SeO3⁻² to insoluble elemental red selenium (Se°). The enzymes associated with this reduction process are poorly understood. In Rhizobium selenitireducens at least two enzymes are potentially involved; one, a nitrite reductase reduces SeO3⁻² to Se° but another protein may also be involved which is investigated in this study. Proteins from R. selenitireducens cells were precipitated with ammonium sulfate and run on native electrophoresis gels. When these gels were incubated with NADH and SeO3⁻² a band of precipitated Se° developed signifying the presence of a SeO3⁻² reducing protein. Bands were cut from the gel and analyzed for peptides via LCMSMS. The amino acid sequences associated with the bands indicated the presence of an NADH:flavin oxidoreductase that resembles YP_001326930 from Sinorhizobium medicae. The protein is part of a protein family termed old-yellow-enzymes (OYE) that contain a flavin binding domain. OYE enzymes are often involved in protecting cells from oxidative stress and, due in part to an active site that has a highly accessible binding pocket, are generally active on a wide range of substrates. This report is the first of an OYE enzyme being involved in SeO3⁻² reduction.

Highly enantioselective oxidation of α-hydroxyacids bearing a substituent with an aryl group: co-production of optically active α-hydroxyacids and α-ketoacids.

A novel and simple methodology for co-obtaining of enantiomerically pure α-hydroxyacids and α-ketoacids was developed by enantioselective oxidation of α-hydroxyacids bearing a substituent with an aryl group using α-hydroxyacid dehydrogenase (α-HADH). A high-throughput method was firstly established for screening of enantioselective α-HADHs. Sinorhizobium sp. ZJB1 101 with high activity and excellent enantioselectivity of α-HADH for oxidation of α-hydroxyacids bearing a substituent with an aryl group was isolated and identified. This strain has potential for co-production of (R)-α-hydroxyacids and α-ketoacids in near theoretical yields, while no consecutive oxidation of α-ketoacids was observed. The green conversion system appears promising for potential applications in industry.

Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method.

To isolate enantioselective α-hydroxyacid dehydrogenases (α-HADHs), a high-throughput screening method was established. 2,4-Dinitrophenylhydrazine solution forms a red-brown complex with ketoacid produced during the α-HADH-mediated oxidation of α-hydroxyacid. The complex can be easily quantified by spectrophotometric measurement at 458 nm. The enantioselectivity of α-HADH in each strain can be measured with this colorimetric method using (R)- and (S)-α-hydroxyacid concurrently as substrates to evaluate the apparent enantioselectivity (E (app)). The E (app) closely matches the value of true enantioselectivity (E (true)) determined by HPLC analysis. With this method, a total of 34 stains harboring enantioselective α-HADHs were selected from 526 potential α-HADH-producing microorganisms. Pseudomonas aeruginosa displayed the highest (S)-enantioselective α-HADH activity. This strain appears promising for potential application in industry to produce (R)-α-hydroxyacids. The method described herein represents a useful tool for the high-throughput isolation of enantioselective α-HADHs.

NAD(P)+-malic enzyme mutants of Sinorhizobium sp. strain NGR234, but not Azorhizobium caulinodans ORS571, maintain symbiotic N2 fixation capabilities.

C(4)-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N(2)-fixing bacteria (bacteroids) within legume nodules. In Sinorhizobium meliloti bacteroids from alfalfa, NAD(+)-malic enzyme (DME) is required for N(2) fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont Rhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that dme mutants of the broad-host-range Sinorhizobium sp. strain NGR234 formed nodules whose level of N(2) fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single pckA and single dme mutants fixed N(2) at reduced rates, a pckA dme double mutant had no N(2)-fixing activity (Fix(-)). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix(-) phenotype of S. meliloti dme mutants may be specific to the alfalfa-S. meliloti symbiosis. We therefore examined the ME-like genes azc3656 and azc0119 from Azorhizobium caulinodans, as azc3656 mutants were previously shown to form Fix(-) nodules on the tropical legume Sesbania rostrata. We found that purified AZC3656 protein is an NAD(P)(+)-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N(2) fixation in A. caulinodans and S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s).

Cloning of the gene encoding alpha-methylserine hydroxymethyltransferase from Aminobacter sp. AJ110403 and Ensifer sp. AJ110404 and characterization of the recombinant enzyme.

Genes encoding alpha-methylserine hydroxymethyltransferase from Aminobacter sp. AJ110403 and Ensifer sp. AJ110404 were cloned and expressed in Escherichia coli. The purified enzymes were homodimers with a 46-kDa subunit and contained 1 mol/mol-subunit of pyridoxal 5'-phosphate. The V(max) of these enzymes catalyzing the conversion of alpha-methyl-L-serine to D-alanine via tetrahydrofolate was 22.1 U/mg (AJ110403) and 15.4 U/mg (AJ110404).

The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala.

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala.

[Recombinant expression of an autoinducer synthase of Sinorhizobium sp.1128 in Escherichia coli].

OBJECTIVE: To explore the roles of quorum sensing system in establishing symbiosis between bacterium Sinorhizobium sp.1128 and its plant host Melilotus suaveolens Ledeb. METHODS: According to homologous analysis, we designed primers to amplify the autoinducer synthase encoding genes in Sinorhizobium sp.1128 according to Sinorhizobium medicae WSM419 genome sequences. The autoinducer synthase encoding genes were cloned into the expression vector of pYC12 and expressed in E. coli DH5alpha. Thin-layer chromatography (TLC) assay was used to study their roles in autoinducer production. A duplicated inactivation of the gene was used to explore its function in plant nodulation. RESULTS: Homologous analysis showed that at least three annotated acylated homoserine lactone (AHL) synthase genes existed in Sinorhizobium medicae WSM419 genome. We cloned these three autoinducer synthase genes in Sinorhizobium sp.1128. One of these genes named traI2 was over expressed in E. coli DH5alpha. At least two different AHLs were produced by the recombinant strain. Disruption of traI2 reduced both the autoinducers (AI) activities and AHL production by TLC detection. Furthermore, the complementation of traI2 reverted the phenotype of AI activities. These findings demonstrate that traI2 was responsible for AI synthesis in Sinorhizobium sp.1128. More important, the traI2 deficient strains were defective in nodule formation on their host plant. CONCLUSION: The quorum sensing circuits in Sinorhizobium sp.1128 may play an important role in symbiosis between plant and bacterium.

Purification and characterization of extracellular beta-glucosidase from Sinorhizobium kostiense AFK-13 and its algal lytic effect on Anabaena flos-aquae.

A beta-glucosidase from the algal lytic bacterium Sinorhizobium kostiense AFK-13, grown in complex media containing cellobiose, was purified to homogeneity by successive ammonium sulfate precipitation, and anion-exchange and gel-filtration chromatographies. The enzyme was shown to be a monomeric protein with an apparent molecular mass of 52 kDa and isoelectric point of approximately 5.4. It was optimally active at pH 6.0 and 40'C and possessed a specific activity of 260.4 U/mg of protein against 4-nitrophenyl-beta-D-glucopyranoside (pNPG). A temperature-stability analysis demonstrated that the enzyme was unstable at 50 degrees C and above. The enzyme did not require divalent cations for activity, and its activity was significantly suppressed by Hg+2 and Ag+, whereas sodium dodecyl sulfate (SDS) and Triton X-100 moderately inhibited the enzyme to under 70% of its initial activity. In an algal lytic activity analysis, the growth of cyanobacteria, such as Anabaena flos-aquae, A. cylindrica, A. macrospora, Oscillatoria sancta, and Microcystis aeruginosa, was strongly inhibited by a treatment of 20 ppm/disc or 30 ppm/disc concentration of the enzyme.

Inactivation of the nodH gene in Sinorhizobium sp. BR816 enhances symbiosis with Phaseolus vulgaris L.

Sulfate modification on Rhizobium Nod factor signaling molecules is not a prerequisite for successful symbiosis with the common bean (Phaseolus vulgaris L.). However, many bean-nodulating rhizobia, including the broad host strain Sinorhizobium sp. BR816, produce sulfated Nod factors. Here, we show that the nodH gene, encoding a sulfotransferase, is responsible for the transfer of sulfate to the Nod factor backbone in Sinorhizobium sp. BR816, as was shown for other rhizobia. Interestingly, inactivation of nodH enables inoculated bean plants to fix significantly more nitrogen under different experimental setups. Our studies show that nodH in the wild-type strain is still expressed during the later stages of symbiosis. This is the first report on enhanced nitrogen fixation by blocking Nod factor sulfation.

[Cloning, expression and purification of D-carbamoylase from Sinorhizobium morelens S-5].

A N-carbamoyl-D-amino acid amidohydrolase gene (hyuC) from Sinorhizobium morelens S-5 was cloned by LA PCR, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the hyuC gene exhibited high homology to the amino acid sequences of D-carbamoylase from other sources. The gene could be highly expressed in Escherichia coli, and the recombinant enzyme was purified 16.1-fold to homogeneity with a yield of 21.2% by heat treatment and three steps of column chromatography. The results of gel filtration on Superdex 200 HR and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 38-kDa subunits. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha-amino acid to the corresponding free amino acid, and it was strictly D-specific. The enzyme showed broad substrate specificity, and exhibited high activity in the hydrolysis of N-carbamoyl-D-p-hydroxyphenylglycine as substrate. The enzyme did not hydrolyze N-carbamoyl-beta-alanine. The optimum pH and temperature of the enzyme were pH 7.0 and 60 degrees C, respectively. Enzyme activity was slightly improved by Ca2+ and Fe2+, and nearly not affected by metal chelators and sulfhydryl reagents. The enzyme showed high thermal and oxidative stability. These results show that the enzyme has great potential for industrial application.

Crystal structure of NADP(H)-dependent 1,5-anhydro-D-fructose reductase from Sinorhizobium morelense at 2.2 A resolution: construction of a NADH-accepting mutant and its application in rare sugar synthesis.

Recombinant 1,5-anhydro-d-fructose reductase (AFR) from Sinorhizobium morelense S-30.7.5 was crystallized in complex with the cofactor NADP(H) and its structure determined to 2.2 A resolution using selenomethionine SAD (refined R(work) and R(free) factors of 18.9 and 25.0%, respectively). As predicted from the sequence and shown by the structure, AFR can be assigned to the GFO/IDH/MocA protein family. AFR consists of two domains. The N-terminal domain displays a Rossmann fold and contains the cofactor binding site. The intact crystals contain the oxidized cofactor NADP(+), whose attachment to the cofactor binding site is similar to that of NADP(+) in glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. Due to variations in length and sequence within loop regions L3 and L5, respectively, the adenine moiety of NADP(+) adopts a different orientation in AFR caused by residue Arg38 forming hydrogen bonds with the 2'-phosphate moiety of NADP(+) and cation-pi stacking interactions with the adenine ring. Amino acid replacements in AFR (S10G, A13G, and S33D) showed that Ala13 is crucial for the discrimination between NADPH and NADH and yielded the A13G variant with dual cosubstrate specificity. The C-terminal domain contains the putative substrate binding site that was occupied by an acetate ion. As determined by analogy to GFOR and by site-directed mutagenesis of K94G, D176A, and H180A, residues Lys94, Asp176, and His180 are most likely involved in substrate binding and catalysis, as substitution of any of these residues resulted in a significant decrease in k(cat) for 1,5-AF. In this context, His180 might serve as a general acid-base catalyst by polarizing the carbonyl function of 1,5-AF to enable the transfer of the hydride from NADPH to the substrate. Here we present the first structure of an AFR enzyme catalyzing the stereoselective reduction of 1,5-AF to 1,5-anhydro-d-mannitol, the final step of a modified anhydrofructose pathway in S. morelense S-30.7.5. We also emphasize the importance of the A13G variant in biocatalysis for the synthesis of 1,5-AM and related derivatives.

Thermostable D-carbamoylase from Sinorhizobium morelens S-5: purification, characterization and gene expression in Escherichia coli.

A d-carbamoylase from Sinorhizobium morelens S-5 was purified and characterized. The enzyme was purified 189-fold to homogeneity with a yield of 19.1% by aqueous two-phase extraction and two steps of column chromatography. The enzyme is a homotetramer with a native molecular mass of 150 kDa and a subunit relative molecular mass of 38 kDa. The optimum pH and temperature of the enzyme were pH 7.0 and 60 degrees C, respectively. The enzyme showed high thermal and oxidative stability. It was found to have a K(m) of 3.76 mM and a V(max) of 383 U/mg for N-carbamoyl-d-p-hydroxyphenylglycine. The hyuC gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the hyuC gene exhibited high homology to the amino acid sequences of d-carbamoylase from other sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity from the recombinant. Our results show that the enzyme has great potential for industrial application.

Nitrous oxide reductase genes (nosZ) of denitrifying microbial populations in soil and the earthworm gut are phylogenetically similar.

Earthworms emit nitrous oxide (N2O) and dinitrogen (N2). It has been hypothesized that the in situ conditions of the earthworm gut activates ingested soil denitrifiers during gut passage and leads to these in vivo emissions (M. A. Horn, A. Schramm, and H. L. Drake, Appl. Environ. Microbiol. 69:1662-1669, 2003). This hypothesis implies that the denitrifiers in the earthworm gut are not endemic to the gut but rather are regular members of the soil denitrifier population. To test this hypothesis, the denitrifier populations of gut and soil from three different sites were comparatively assessed by sequence analysis of nosZ, the gene for the terminal enzyme in denitrification, N2O reductase. A total of 182 and 180 nosZ sequences were retrieved from gut and soil, respectively; coverage of gene libraries was 79 to 100%. Many of the nosZ sequences were heretofore unknown, clustered with known soil-derived sequences, or were related to N2O reductases of the genera Bradyrhizobium, Brucella, Dechloromonas, Flavobacterium, Pseudomonas, Ralstonia, and Sinorhizobium. Although the numbers of estimators for genotype richness of sequence data from the gut were higher than those of soil, only one gut-derived nosZ sequence did not group phylogenetically with any of the soil-derived nosZ sequences. Thus, the phylogenies of nosZ from gut and soil were not dissimilar, indicating that gut denitrifiers are soil derived.

Catabolism of 1,5-anhydro-D-fructose in Sinorhizobium morelense S-30.7.5: discovery, characterization, and overexpression of a new 1,5-anhydro-D-fructose reductase and its application in sugar analysis and rare sugar synthesis.

The bacterium Sinorhizobium morelense S-30.7.5 was isolated by a microbial screening using the sugar 1,5-anhydro-D-fructose (AF) as the sole carbon source. This strain metabolized AF by a novel pathway involving its reduction to 1,5-anhydro-D-mannitol (AM) and the further conversion of AM to D-mannose by C-1 oxygenation. Growth studies showed that the AF metabolizing capability is not confined to S. morelense S-30.7.5 but is a more common feature among the Rhizobiaceae. The AF reducing enzyme was purified and characterized as a new NADPH-dependent monomeric reductase (AFR, EC 1.1.1.-) of 35.1 kDa. It catalyzed the stereoselective reduction of AF to AM and also the conversion of a number of 2-keto aldoses (osones) to the corresponding manno-configurated aldoses. In contrast, common aldoses and ketoses, as well as nonsugar aldehydes and ketones, were not reduced. A database search using the N-terminal AFR sequence retrieved a putative 35-kDa oxidoreductase encoded by the open reading frame Smc04400 localized on the chromosome of Sinorhizobium meliloti 1021. Based on sequence information for this locus, the afr gene was cloned from S. morelense S-30.7.5 and overexpressed in Escherichia coli. In addition to the oxidoreductase of S. meliloti 1021, AFR showed high sequence similarities to putative oxidoreductases of Mesorhizobium loti, Brucella suis, and B. melitensis but not to any oxidoreductase with known functions. AFR could be assigned to the GFO/IDH/MocA family on the basis of highly conserved common structural features. His6-tagged AFR was used to demonstrate the utility of this enzyme for AF analysis and synthesis of AM, as well as related derivatives.