Editor\u27s Notes, Chiba Medical Journal 90-1

Multiple sequence alignment of deduced amino acid sequences of 25 wheat annexin genes with rice annexin OsAnn2 (Os05g31760) obtained by ClustalW. (PDF 212 kb

Characterization of Protein Encoded by <i>spnR</i> from the Spinosyn Gene Cluster of <i>Saccharopolyspora </i><i>s</i><i>pinosa</i>: Mechanistic Implications for Forosamine Biosynthesis

d-Forosamine is a 4-N,N-(dimethylamino)-2,3,4,6-tetradeoxy-α-d-threo-hexopyranose found in spinosyn produced by Saccharopolyspora spinosa. Studies of spinosyn biosynthesis in S. spinosa led to the isolation of the entire biosynthetic gene cluster. Heterologous expression of spnR, one putative gene in forosamine biosynthesis, in E. coli and purification of the SpnR protein identified it as an aminotransferase catalyzing the conversion of the 4-keto-2,3,6-trideoxy sugar intermediate to the corresponding 4-amino sugar product. Identification of SpnR function relied on the use of a stable TMP-phosphonate sugar in place of TDP-sugar substrate to determine the function of SpnR. This strategy may find general applicability for designing probes to study enzymes which catalyze the transformation of labile deoxysugar intermediates

Characterization of SpnQ from the Spinosyn Biosynthetic Pathway of <i>Saccharopolyspora spinosa</i>: Mechanistic and Evolutionary Implications for C-3 Deoxygenation in Deoxysugar Biosynthesis

The C-3 deoxygenation step in the biosynthesis of d-forosamine (4-N,N-dimethylamino-2,3,4,6-tetradeoxy-d-threo-hexopyranose), a constituent of spinosyn produced by Saccharopolyspora spinosa, was investigated. The spnQ gene, proposed to encode a TDP-4-keto-2,6-dideoxy-d-glucose 3-dehydratase was cloned and overexpressed in E. coli. Characterization of the purified enzyme established that it is a PMP and iron-sulfur containing enzyme which catalyzes the C-3 deoxygenation in a reductase-dependent manner similar to that of the previously well characterized hexose 3-dehydrase E1 from Yersinia pseudotuberculosis. However, unlike E1, which has evolved to work with a specific reductase partner present in its gene cluster, SpnQ lacks a specific reductase, and works efficiently with general cellular reductases ferredoxin/ferredoxin reductase or flavodoxin/flavodoxin reductase. SpnQ also catalyzes C-4 transamination in the absence of an electron transfer intermediary and in the presence of PLP and l-glutamate. Under the same conditions, both E1 and the related hexose 3-dehydrase, ColD, catalyze C-3 deoxygenation. Thus, SpnQ possesses important features which distinguish it from other well studied homologues, suggesting unique evolutionary pathways for each of the three hexose 3-dehydrases studied thus far

Main stages in the continuous filling of micropores during the adsorption process.

Main stages in the continuous filling of micropores during the adsorption process.</p

Curvature curves of the adsorption isotherms of coal samples of different coal ranks.

Curvature curves of the adsorption isotherms of coal samples of different coal ranks.</p

Cumulative pore volume and differential pore volume diagram of coals.

(a) PH Sample, (b) JB Sample, (c) LH Sample, (d) HG Sample, (e) CV Sample, (f) YW Sample.</p

Adsorption/desorption isotherms of coal samples of different coal ranks.

(a) PH Sample, (b) JB Sample, (c) LH Sample, (d) HG Sample, (e) CV Sample, (f) YW Sample.By analyzing the types of hysteresis loops of the six coal samples, the N2 adsorption/desorption isotherm of the PH coal sample can be approximated to H2-type hysteresis loops, indicating that the coal samples may contain some ink bottle holes. The JB, LH, and HG coal samples belong to H3-type hysteresis loops, indicating that the coal samples are mainly composed of non-uniform slit holes formed by the accumulation of flake particles. The CV and YW coal samples belong to H4-type hysteresis loops, similar in shape to H3-type. However, samples showing H4-type typically contain uniform and narrow slit pores, indicating the presence of non-rigid aggregates of micropores and flaky particles in the coal sample. Except for the PH coal sample, the desorption branch of the other five coal samples suddenly dropped when P/P0 = 0.42–0.5, approaching the adsorption branch; however, there was no closure point in the adsorption/desorption isotherm hysteresis loop. This phenomenon is typically due to solid swelling (intercalation phenomenon) [28] or due to the nature of the sample itself. The desorption branch deviations of the CV and YW coal samples were more evident than those of the other coal samples. This is attributed to the expansion of the interlayer distance due to adsorption. The interlayer distance is several times the molecular diameter, which is close to the micropore size, and the interlayer is then entered. The gas molecules are difficult to desorb; therefore, the isotherm does not close even under a very low relative pressure.</p

Experimental flowchart.

Experimental flowchart.</p

Differential curves of the adsorption isotherms of coal samples of different coal ranks.

Differential curves of the adsorption isotherms of coal samples of different coal ranks.</p

Fitting diagrams of coal samples of different coal ranks obtained using different models at different pressure sections.

(a) PH Sample, (b) JB Sample, (c) LH Sample. (Left: Langmuir and DA model, Right: Langmuir, DA, and BET models).</p