4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. A novel flavoprotein catalyzing Baeyer-Villiger oxidation of aromatic compounds.

A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-hydroxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4-hydroxyacetophenone monooxygenase (HAPMO) is a homodimer of approximately 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4-hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer-Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino-acid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of approximately 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer-Villiger monooxygenases: cyclohexanone monooxygenase (27-33% sequence identity) and steroid monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site-directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.

Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes.

Pseudomonas sp. GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is toxic at low concentrations (1 mM). A mutant that could grow on 2-bromoethanol with a growth rate of 0.034 h-1 at concentrations up to 5 mM was isolated and designated strain GJ1M9. Measurement of enzyme activities showed that mutant and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was threefold overexpressed in the mutant when grown on 2-chloroethanol, but only slightly overproduced when grown on 2-bromoethanol. Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols. Haloacetate dehalogenase levels were similar in the wild-type and the mutant. Activities of NAD-dependent aldehyde dehydrogenase were only slightly higher in extracts of the mutant grown with 2-bromoethanol than in those of the wild-type grown with 2-chloroethanol. SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2-chloroethanol. The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa. The enzyme had low Km values for acetaldehyde and other non-halogenated aldehydes (0.8-4 microM), but much higher Km values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while V(max) values were similar for halogenated and non-halogenated aldehydes. Cultures that were pregrown on 2-chloroethanol rapidly lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde inactivates the enzyme. To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary in order to compensate for the high Km for bromoacetaldehyde and for inactivation of the enzyme of bromoacetaldehyde.

Synthesis of a fucosylated and a non-fucosylated core structure of xylose-containing carbohydrate chains from N-glycoproteins.

The synthesis is reported of methyl 2-acetamido-4-O-[2-acetamido-2-deoxy-O-(3,6-di-O-alpha-D- mannopyranosyl-2-O-beta-D-xylopyranosyl-beta-D-mannopyranosyl)-beta-D- glucopyranosyl]-2-deoxy-beta-D-glucopyranoside (4) and methyl 2-acetamido-4-O-[2-acetamido-2-deoxy-4-O- (3,6-di-O-alpha-D- mannopyranosyl-2-O-beta-D-xylopyranosyl-beta-D-mannopyranosyl)-beta-D- glucopyranosyl]-2-deoxy-6- O-alpha-L-fucopyranosyl-beta-D-glucopyranoside (5), which represent the invariant hexasaccharide core structure of the xylose-containing glycans of N-glycoproteins and its 6-O- fucosylated derivative. Ethyl 4-O-[3-O-allyl-4-O-benzoyl-6-O-tert-butyldimethylsilyl-2-O- (2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1- thio-beta-D-glucopyranoside (9) was coupled with methyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranoside (11). Desilylation of the resulting tetrasaccharide derivative, followed by condensation with 2,3,4,6- tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (7), gave methyl 4-O-(4-O-[3-O-allyl-4- O-benzoyl-6-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-2-O-(2,3,4 -tri-O- acetyl-beta-D-xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)- 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside (14). Deallylation of 14, followed by condensation with 7 and deprotection, gave hexasaccharide 4. Ethyl 3,6-di-O- benzyl-2-deoxy-4-O- [4,6-di-O-acetyl-3-O-allyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl) - beta-D-mannopyranosyl]-2- phthalimido-1-thio-beta-D-glucopyranoside (17) was coupled with methyl 3-O- benzyl-2-deoxy-6-O- (4-methoxybenzyl)-2-phthalimido-beta-D-glucopyranoside. Demethoxybenzylation of the tetrasaccharide derivative thus obtained, followed by fucosylation using ethyl 2,3,4-tri-O- benzyl-1-thio- beta-L-fucopyranoside, gave methyl 3-O-benzyl-2-deoxy-4-O-[3,6-di-O-benzyl-2- deoxy-4-O-[4,6- di-O-acetyl-3-O-allyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta -D- mannopyranosyl]-2-phthalimido- beta-D-glucopyranosyl)-2-phthalimido-6-O-(2,3,4-tri-O-benzyl-alpha-L- fucopyranosyl)-beta-D-glucopyranoside (23). O-Deacetylation followed by tert-butyldimethylsilylation, benzoylation, and desilylation gave methyl 4-O-(4-O-[3-O-allyl-4-O-benzoyl-2-O-(2,3,4-tri-O-benzoyl-beta-D- xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta- D-glucopyranosyl)-3- O-benzyl-2-deoxy-2-phthalimido-6-O-(2,3,4-tri-O-benzyl-alpha-L-fucopy ran osyl)- beta-D-glucopyranoside (24). Mannosylation of 24 using 7, followed by deallylation, further mannosylation with 7, and deprotection, gave the heptasaccharide 5.

Synthesis of three tetrasaccharides containing 3-O-methyl-D-mannose, as model compounds for xylose-containing carbohydrate chains from N-glycoproteins.

The synthesis is reported of methyl 3,6-di-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-beta-D-xylopyranosyl-be ta-D- mannopyranoside (2), methyl 6-O-alpha-D-mannopyranosyl-3-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-b eta-D- xylopyranosyl-beta-D-mannopyranoside (3), and methyl 3-O-alpha-D-mannopyranosyl-6-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-b eta-D- xylopyranosyl-beta-D-mannopyranoside (4). The various methyl beta-D-Manp acceptor derivatives were prepared from the corresponding methyl beta-D-Glcp derivatives via oxidation-reduction. All glycosyl donors were coupled using the trichloroacetimidate method at -40 degrees C in dichloromethane with trimethylsilyl triflate as a catalyst. Methyl-3-O-benzyl-4,6-O-benzylidene-beta-D-mannopyranoside (7) was condensed with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl trichloroacetimidate (8). Regioselective reductive 4,6-O-benzylidene ring-opening on the resulting disaccharide derivative, followed by acetylation, and hydrogenation gave methyl 4-O-acetyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta-D-mannopyr anoside (12). Coupling of 12 with 2,4,6-tri-O-acetyl-3-O-methyl-alpha-D-mannopyranosyl trichloroacetimidate (18) afforded tetrasaccharide derivative 19, and subsequent O-deacetylation gave 2. Methyl 3-O-benzyl-4,6-O-prop-2-enylidene-beta-D-mannopyranoside (22) was condensed with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl trichloroacetimidate (8). Regioselective reductive 4,6-O-prop-2-enylidene ring-opening on the resulting disaccharide derivative, followed by acetylation, and deallylation at O-6 gave methyl 4-O-acetyl-3-O-benzyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta -D- mannopyranoside (26-a), which was either condensed with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (27) or 18, to give trisaccharide derivatives 28 or 31, respectively. Debenzylation of 28 followed by condensation with 18 gave, after O-deacetylation, 3, whereas debenzylation of 31 followed by condensation with 27 gave, after O-deacetylation, 4.

Action of rat liver Gal beta 1-4GlcNAc alpha(2-6)-sialyltransferase on Man beta 1-4GlcNAc beta-OMe, GalNAc beta 1-4GlcNAc beta-OMe, Glc beta 1-4GlcNAc beta-OMe and GlcNAc beta 1-4GlcNAc beta-OMe as synthetic substrates.

Incubation of synthetic Man beta 1-4GlcNAc beta-OMe, GalNAc beta 1-4GlcNAc beta-OMe, Glc beta 1-4GlcNAc beta-OMe, and GlcNAc beta 1-4GlcNAc beta-OMe with CMP-Neu5Ac and rat liver Gal beta 1-4GlcNAc alpha(2-6)-sialyltransferase resulted in the formation of Neu5Ac alpha 2-6Man beta 1-4GlcNAc beta-OMe, Neu5Ac alpha 2-6GalNAc beta 1-4GlcNAc beta-OMe, Neu5Ac alpha 2-6Glc beta 1-4GlcNAc beta-OMe and Neu5Ac alpha 2-6GlcNAc beta 1-4GlcNAc beta-OMe, respectively. Under conditions which led to quantitative conversion of Gal beta 1-4GlcNAc beta-OEt into Neu5Ac alpha 2-6Gal beta 1-4GlcNAc beta-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz 1H-NMR spectroscopy.

Synthesis of a selectively protected trisaccharide building block that is part of xylose-containing carbohydrate chains from N-glycoproteins.

The synthesis is reported of ethyl 4-O-[3-O-allyl-4,6-O-isopropylidene-2-O-(2,3,4-tri-O-acetyl-beta-D- xylopyranosyl)-beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalim ido-1 - thio-beta-D-glucopyranoside (16), a key intermediate in the synthesis of xylose-containing carbohydrate chains from N-glycoproteins. Condensation of ethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D- glucopyranoside (5) with 2,4,6-tri-O-acetyl-3-O-allyl-alpha-D-glucopyranosyl bromide, using silver triflate as a promoter, gave the beta-linked disaccharide derivative 8 (84%). O-Deacetylation of 8 and then isopropylidenation afforded 10, which was converted via oxidation-reduction into ethyl 4-O-(3-O-allyl-4,6-O-isopropylidene-beta-D-mannopyranosyl)-3,6-di-O-benz yl-2- deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (12). Silver triflate-promoted condensation of 12 with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl bromide gave 16 (71%). The Xylp unit in 16 and in de-isopropylidenated 16 (17) existed in the 1C4(D) conformation, but that in O-deacetylated 17 (18) existed in the 4C1(D) conformation.

Identification of a novel UDP-Gal:GalNAc beta 1-4GlcNAc-R beta 1-3-galactosyltransferase in the connective tissue of the snail Lymnaea stagnalis.

Connective tissue of the freshwater pulmonate Lymnaea stagnalis was shown to contain galactosyltransferase activity capable of transferring Gal from UDP-Gal in beta 1-3 linkage to terminal GalNAc of GalNAc beta 1-4GlcNAc-R [R = beta 1-2Man alpha 1-O(CH2)8COOMe, beta 1-OMe, or alpha,beta 1-OH]. Using GalNAc beta 1-4GlcNAc beta 1-2Man alpha-1-O(CH2)8COOMe as substrate, the enzyme showed an absolute requirement for Mn2+ with an optimum Mn2+ concentration between 12.5 mM and 25 mM. The divalent cations Mg2+, Ca2+, Ba2+ and Cd2+ at 12.5 mM could not substitute for Mn2+. The galactosyltransferase activity was independent of the concentration of Triton X-100, and no activation effect was found. The enzyme was active with GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOMe (Vmax 140 nmol.h-1.mg protein-1; Km 1.02 mM), GalNAc beta 1-4GlcNAc (Vmax 105 nmol.h-1.mg protein-1; Km 0.99 mM), and GalNAc beta 1-4GlcNAc beta 1-OMe (Vmax 108 nmol.h-1.mg protein-1; Km 1.33 mM). The products formed from GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOMe and GalNAc beta 1-4GlcNAc beta 1-OMe were purified by high performance liquid chromatography, and identified by 500-MHz 1H-NMR spectroscopy to be Gal beta 1-3GalNAc beta 1-4GlcNAc 1-OMe, respectively. The enzyme was inactive towards GlcNAc, GalNac beta 1-3 GalNAc alpha 1-OC6H5, GalNAc alpha 1--ovine-submaxillary-mucin, lactose and N-acetyllactosamine. This novel UDP-Gal:GalNAc beta 1-4GlcNAc-R beta 1-3-galactosyltransferase is believed to be involved in the biosynthesis of the hemocyanin glycans of L. stagnalis.