Thesis (M. Phil.)--University of Hong Kong, 1974. / Mimeographed.

Thesis (Ph. D.)--University of Hong Kong, 1982.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

ADP-L-glycero-D-manno-heptose 6-epimerase (HldD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. H1dD operates in the biosynthesis of L-glycero-D-manno-heptose, a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. This work supports a direct redox mechanism whereby H1dD uses its tightly bound NADP+ to oxidize the substrate at C-6", generating a ketone intermediate. Reduction from the opposite face generates the epimeric product. An analog of the ketone intermediate, ADP-ß-D-manno-hexodialdose 8, was shown to undergo dismutation giving equal amounts of ADP-mannose 9and ADP-mannuronate 10. Observation of transient NADPH during dismutation established participation of the tightly bound cofactor. Further studies address how HldD is able to access both faces of the ketone intermediate with correct alignment of NADPH, the ketone intermediate, and a catalytic acid/base residue. It is proposed that Escherichia coli K-12 HldD contains two catalytic acid/base residues, tyrosine 140 and lysine 178, each of which facilitates redox chemistry on opposite faces of the ketone intermediate. The ketone intermediate may access either base via rotation about the C-5"/C-6" bond. The observation that two single mutants, Y140F and K178M, have severely compromised epimerase activities, yet retain dismutase activity, supports this hypothesis. UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5" stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetyl-2,6-dideoxy-ß-L-arabino-4-hexulose. PseB catalyses the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylon. PseB uses its tightly bound NADP+ to oxidize UDP-G1cNAc at C-4", enabling dehydration. The a,ß unsaturated ketone intermediate thus generated is reduced by delivery of a hydride from NADPH to C-6", and a proton to C-5". Consistent with this mechanism, a solvent derived deuterium becomes incorporated into the C-5" position of product during catalysis in D20. Likewise, PseB catalyzes solvent isotope exchange into the H5" position of the product, and theelimination of HF from UDP-6-deoxy-6-fluoro-G1cNAc 23. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.

ADP-L-glycero-D-manno-heptose 6-epimerase (HldD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. H1dD operates in the biosynthesis of L-glycero-D-manno-heptose, a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. This work supports a direct redox mechanism whereby H1dD uses its tightly bound NADP+ to oxidize the substrate at C-6", generating a ketone intermediate. Reduction from the opposite face generates the epimeric product. An analog of the ketone intermediate, ADP-ß-D-manno-hexodialdose 8, was shown to undergo dismutation giving equal amounts of ADP-mannose 9and ADP-mannuronate 10. Observation of transient NADPH during dismutation established participation of the tightly bound cofactor. Further studies address how HldD is able to access both faces of the ketone intermediate with correct alignment of NADPH, the ketone intermediate, and a catalytic acid/base residue. It is proposed that Escherichia coli K-12 HldD contains two catalytic acid/base residues, tyrosine 140 and lysine 178, each of which facilitates redox chemistry on opposite faces of the ketone intermediate. The ketone intermediate may access either base via rotation about the C-5"/C-6" bond. The observation that two single mutants, Y140F and K178M, have severely compromised epimerase activities, yet retain dismutase activity, supports this hypothesis. UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5" stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetyl-2,6-dideoxy-ß-L-arabino-4-hexulose. PseB catalyses the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylon. PseB uses its tightly bound NADP+ to oxidize UDP-G1cNAc at C-4", enabling dehydration. The a,ß unsaturated ketone intermediate thus generated is reduced by delivery of a hydride from NADPH to C-6", and a proton to C-5". Consistent with this mechanism, a solvent derived deuterium becomes incorporated into the C-5" position of product during catalysis in D20. Likewise, PseB catalyzes solvent isotope exchange into the H5" position of the product, and theelimination of HF from UDP-6-deoxy-6-fluoro-G1cNAc 23. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.

ADP-L-glycero-D-manno-heptose 6-epimerase (HldD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. H1dD operates in the biosynthesis of L-glycero-D-manno-heptose, a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. This work supports a direct redox mechanism whereby H1dD uses its tightly bound NADP+ to oxidize the substrate at C-6", generating a ketone intermediate. Reduction from the opposite face generates the epimeric product. An analog of the ketone intermediate, ADP-ß-D-manno-hexodialdose 8, was shown to undergo dismutation giving equal amounts of ADP-mannose 9and ADP-mannuronate 10. Observation of transient NADPH during dismutation established participation of the tightly bound cofactor. Further studies address how HldD is able to access both faces of the ketone intermediate with correct alignment of NADPH, the ketone intermediate, and a catalytic acid/base residue. It is proposed that Escherichia coli K-12 HldD contains two catalytic acid/base residues, tyrosine 140 and lysine 178, each of which facilitates redox chemistry on opposite faces of the ketone intermediate. The ketone intermediate may access either base via rotation about the C-5"/C-6" bond. The observation that two single mutants, Y140F and K178M, have severely compromised epimerase activities, yet retain dismutase activity, supports this hypothesis. UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5" stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetyl-2,6-dideoxy-ß-L-arabino-4-hexulose. PseB catalyses the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylon. PseB uses its tightly bound NADP+ to oxidize UDP-G1cNAc at C-4", enabling dehydration. The a,ß unsaturated ketone intermediate thus generated is reduced by delivery of a hydride from NADPH to C-6", and a proton to C-5". Consistent with this mechanism, a solvent derived deuterium becomes incorporated into the C-5" position of product during catalysis in D20. Likewise, PseB catalyzes solvent isotope exchange into the H5" position of the product, and theelimination of HF from UDP-6-deoxy-6-fluoro-G1cNAc 23. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities. / Science, Faculty of / Chemistry, Department of / Graduate

published_or_final_version / Biochemistry / Master / Master of Philosophy

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

No description available.

In the first part of this thesis the metabolism of the phytotoxins destruxin B and sirodesmin PL in crucifers and non-crucifers was studied using HPLC-ESI-MSn. Destruxin B and sirodesmin PL are phytotoxins produced by the phytopathogenic fungi Alternaria brassicae (Berk.) Sacc. (causative agent of blackspot disease) and Leptosphaeria maculans (Desm) Ces. et de Not.[asexual stage Phoma lingam (Tode ex Fr) Desm.] (causative agent of blackleg disease). Five cruciferous species were used in this study: Arabidopsis thaliana L., Brassica rapa L., B. napus L., Thellungiella salsuginea Pallas and Erucastrum gallicum O.E. Schulz. In addition, the cereals Avena sativa L. and Triticum aestivum L. were studied similarly. Destruxin B was metabolized by all crucifers to hydroxydestruxin B, a transformation similar to previously reported reactions in other crucifers. In addition, destruxin B elicited production of phytoalexins in A. thaliana, T. salsuginea and E. gallicum, while no phytoalexins were detected in case of B. rapa and B. napus. In cereals destruxin B was transformed differently. Several metabolites were detected and identified by HPLC-ESI-MSn analyses: hydroxydestruxin B, two isomers of dehydrodestruxin B and desmethyldestruxin B. On the other hand, no metabolites related to transformation of sirodesmin PL were detected in crucifers; however, in cereals sirodesmin PL was transformed to deacetylsirodesmin PL. In all crucifers sirodesmin PL was found to be a stronger elicitor of phytoalexin production than destruxin B. In the second part of this thesis, mycelia from different pathogenic fungi were screened for indolyl-3-acetaldoxime dehydratase. L. maculans isolate Laird 2 was chosen for isolation, characterization and substrate specificity of aldoxime dehydratase, as it showed the highest specific activity among the tested pathogens. The enzyme was partially purified using three chromatographic steps. It showed Michaelis–Menten kinetics and an apparent molecular mass of about 40 kDa. Based on its substrate specificity, the enzyme appears to be an indolyl-3-acetaldoxime dehydratase