L-rhamnulose 1-phosphate Aldolase Research Articles

Chemoenzymatic synthesis, structural study and biological activity of novel indolizidine and quinolizidine iminocyclitols

The synthesis, conformational study and inhibitory properties of diverse indolizidine and quinolizidine iminocyclitols are described. The compounds were chemo-enzymatically synthesized by two-step aldol addition and reductive amination reactions. The aldol addition of dihydroxyacetone phosphate (DHAP) to N-Cbz-piperidine carbaldehyde derivatives catalyzed by L-rhamnulose 1-phosphate aldolase from Escherichia coli provides the key intermediates. The stereochemical outcome of both aldol addition and reductive amination depended upon the structure of the starting material and intermediates. The combination of both reactions furnished five indolizidine and six quinolizidine type iminocyclitols. A structural analysis by NMR and in silico density functional theory (DFT) calculations allowed us to determine the population of stereoisomers with the trans or cis ring fusion, as a consequence of the inversion of configuration of the bridgehead nitrogen. The trans fusion was by far the most stable, but for certain stereochemical configurations of the 3-hydroxymethyl and hydroxyl substituents both trans and cis fusion stereoisomers coexisted in different proportions. Some of the polyhydroxylated indolizidines and quinolizidines were shown to be moderate to good inhibitors against α-L-rhamnosidase from Penicillium decumbens. Indolizidines were found to be moderate inhibitors of the rat intestinal sucrase and of the exoglucosidase amyloglucosidase from Aspergillus niger. In spite of their activity against α-L-rhamnosidase, all the compounds were ineffective to inhibit the growth of the Mycobacterium tuberculosis, the causative agent of tuberculosis.

アルドラーゼを用いた糖類合成の展開

Facile chemoenzymatic methods for the synthesis of a variety of D- and L- iminocyclitols or L-sugars have been developed. The practicality of formerly reported methods using dihydroxyacetone phosphate (DHAP) aldolase was limited by the high cost and instability of DHAP. Here we discuss three strategies toward the facile synthesis of sugar analogues using DHA(P) aldolases from readily available non-phosphoryrated donor substrates. (1) Directed evolution of the L-rhamnulose 1-phosphate aldolase (RhaD) was employed to alter the donor substrate specificity of RhaD aldolase from DHAP to DHA. In vivo selection for the directed evolution using genetically engineered E. coli strain was constructed . (2) RhaD aldolase was found to accept non-phosphorylated dihydroxyacetone (DHA) as a donor substrate in the presence of borate. We applied this discovery to develop a practical one-step synthesis of L-fructose and two-step synthesis of L-iminocyclitols . (3) A one-pot synthesis was achieved using the recently discovered D-fructose 6-phosphate aldolse (FSA). FSA utilized DHA, hydroxyacetone, and 1-hydroxy-2-butanone as donor substrates to allow the synthesis of a variety of novel D-iminocyclitols in a concise fashion.

Quo vadis photorespiration: A tale of two aldolases

An O 2-consuming side reaction of d-ribulose 1,5-bisphosphate carboxylase causes photorespiration in plants. This reaction may be an inevitable consequence of the enzyme's inability to protect its ene-diolate reaction intermediate from O 2, a notion that is supported by the failure of persistent efforts to eliminate selectively its oxygenase activity by genetic manipulation. We have examined two aldolases with similar ene-diolate intermediates, l-rhamnulose 1-phosphate aldolase and l-fuculose 1-phosphate aldolase. The former enzyme has an oxygenase activity, while the latter does not, suggesting that the reaction with O 2 is not inevitable.

A chemoenzymic synthesis of 1,5-dideoxy-1,5-imino- l-mannitol and l-rhamnitol and investigation of their effects on glycosidases

1,5-Dideoxy-1,5-imino- l-rhamnitol ( 3) and 1,5-dideoxy-1,5-imino- l-mannitol ( 2), designed as inhibitors of α- l-rhamnosidase, have been synthesised from dihydroxyacetone phosphate and 3-azido-2-hydroxypropanal with the acid of partially purified E. coli l-rhamnulose 1-phosphate aldolase free of d-fructose 1,6-bisphosphate aldolase activity. Inhibitory effects of compounds 2, 3, 1,5-dideoxy-1,5-imino- d-mannitol, and 1,5-dideoxy-1,5-imino- d-glucitol ( 12) were tested on selected glycosidases. It has been found that compound 3 is a good inhibitor of α- l-rhamnosidase as well as α- l-fucosidases from bovine kidney and epididymis, 2 is a potent inhibitor of α-galactosidase and a moderate inhibitor of α- l-rhamnosidase, and 12 is a good inhibitor of α-galactosidase and α- l-rhamnosidase.

Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli.

Dissimilation of L-fucose as a carbon and energy source by Escherichia coli involves a permease, an isomerase, a kinase, and an aldolase encoded by the fuc regulon at minute 60.2. Utilization of L-rhamnose involves a similar set of proteins encoded by the rha operon at minute 87.7. Both pathways lead to the formation of L-lactaldehyde and dihydroxyacetone phosphate. A common NAD-linked oxidoreductase encoded by fucO serves to reduce L-lactaldehyde to L-1,2-propanediol under anaerobic growth conditions, irrespective of whether the aldehyde is derived from fucose or rhamnose. In this study it was shown that anaerobic growth on rhamnose induces expression of not only the fucO gene but also the entire fuc regulon. Rhamnose is unable to induce the fuc genes in mutants defective in rhaA (encoding L-rhamnose isomerase), rhaB (encoding L-rhamnulose kinase), rhaD (encoding L-rhamnulose 1-phosphate aldolase), rhaR (encoding the positive regulator for the rha structural genes), or fucR (encoding the positive for the fuc regulon). Thus, cross-induction of the L-fucose enzymes by rhamnose requires formation of L-lactaldehyde; either the aldehyde itself or the L-fuculose 1-phosphate (known to be an effector) formed from it then interacts with the fucR-encoded protein to induce the fuc regulon.

19] l-Rhamnulose 1-Phosphate

Publisher Summary This chapter describes the preparation, purification, and crystallization of L-Rhamnulose 1-phosphate. L-Rhamnulose 1-phosphate may be prepared by using two mutant strains of Escherichia coli. E. coli K12 HfrP72 is grown overnight with shaking at 37° in Difco nutrient broth to the stationary phase. The mutagen N-methyl-N-nitroso-N'-nitroguanidine is added to a final concentration of 80 μg/ml, and the culture is held without shaking at 37° for 1 hr. L-Rhamnulose 1-phosphate aldolase is detected, after removal of nucleoproteins with MnCl2, with a coupled assay system. Two such mutants isolated used in the preparation described are: Rha-58 (kinaseless) and Rha-54 (aldolaseless). They are maintained on nutrient agar slants. The preparation of L-Rhamnulose is discussed. In the preparation of L-Rhamnulose1-phosphate, cells of the E. coli strain Rha -54 from 10 liters of culture medium are suspended in 75 ml of 20 mM potassium-phosphate buffer, pH 7.0, and disrupted in the French pressure cell. The supernatant fluid is concentrated to about 12 ml at 37° and chromatographed on a Sephadex G-10 column prepared and eluted with distilled water. Fractions containing L-rhamnulose 1-phosphate, detected by paper chromatography in butanone:acetic acid:water (15:5:2 v/v/v) followed by molybdic acid spray, are pooled and concentrated at 37°. L-Rhamnulose 1-phosphate, located by spraying a guide strip, is eluted from the paper with water, concentrated, rechromatographed in the same way, eluted with water, and concentrated at 37° to 10 ml. The preparation of crystalline dicyclohexylammonium salt of L-rhamnulose 1-phosphate is also discussed.

L-Rhamnulose 1-phosphate aldolase of Escherichia coli. Role of metal in enzyme structure

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTL-Rhamnulose 1-phosphate aldolase of Escherichia coli. Role of metal in enzyme structureNancy B. Schwartz, Dinah Abram, and David S. FeingoldCite this: Biochemistry 1974, 13, 8, 1726–1730Publication Date (Print):April 1, 1974Publication History Published online1 May 2002Published inissue 1 April 1974https://doi.org/10.1021/bi00705a026RIGHTS & PERMISSIONSArticle Views62Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

L-Rhamnulose 1-phosphate aldolase from Escherichia coli. III. The role of divalent cations in enzyme activity

l-Rhamnulose 1-phosphate aldolase is a zinc metalloenzyme which contains 2 g-atoms of zinc per mole of enzyme [Ref. 2]. A study has been made of the effect of the metal chelator 1,10 phenanthroline on the reversible cleavage of l-rhamnulose 1-P catalyzed by the aldolase. 1,10 phenanthroline is an inhibitor strictly competitive with l-rhamnulose 1-phosphate or dihydroxyacetone phosphate (DHAP), indicating involvement of zinc at the binding site of these compounds. It is a noncompetitive inhibitor with l-lactaldehyde, suggesting that l-lactaldehyde binds to a site on the enzyme not identical to that for ketose phosphate. DHAP, but not l-lactaldehyde prevents loss of zinc from the enzyme during dialysis against chelators at pH 5.0. Native l-rhamnulose 1-phosphate aldolase and succinylated aldolase were shown by equilibrium dialysis to bind 2 moles of DHAP per mole enzyme. Metal-free subunits of the enzyme, or enzyme inactivated by treatment with 8-hydroxyquinoline 5-sulfonic acid or p-mercuribenzoic acid binds less than 1 mole of DHAP per mole of enzyme. The data suggest that each molecule of enzyme has two binding sites for DHAP and that the metal and sulfhydryl integrity of the enzyme must be maintained for binding and catalysis to occur.

L-Rhamnulose 1-phosphate aldolase from Escherichia Coli. II. Characterization as a zinc metalloenzyme

l-Rhamnulose 1-phosphate aldolase ( l-rhamnulose 1-phosphate l-lactaldehyde lyase, EC 4.1.2.b) was sensitive to inhibition by the metal chelators, 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, 1,10-phenanthroline, and 2,2-bipyridine. The relative effectiveness of the inhibition paralleled the ability of the chelators to bind zinc. The activity of inhibited enzyme was restored by Zn(II), Co(II), Ni(II), and Mn(II). Fe(II), Mg(II), Ca(II), and Cu(II) did not restore activity. Atomic absorption analysis established the presence of 2 g-atoms of Zn per mole of native enzyme (mol wt 135,000). No significant amounts of metals other than Zn were observed in the native enzyme. The data suggest that l-rhamnulose 1-P aldolase is a metalloenzyme and that zinc is required for activity.

Substrate specificity of L-rhamnulose 1-phosphate aldolase

Substrate specificity of L-rhamnulose 1-phosphate aldolase