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studies of the biosynthesis of bacterial cell wall and antibiotic deoxysugar moieties: dtdp-rhamnose, cdp-ascarylose, and dtdp-mycaminose (yersinia pseudotuberculosis, streptomyces fradiae). [microform]
studies of the biosynthesis of bacterial cell wall and antibiotic deoxysugar moieties: dtdp-rhamnose, cdp-ascarylose, and dtdp-mycaminose (yersinia pseudotuberculosis, streptomyces fradiae). [microform]
상세정보
- 자료유형
- 마이크로피시
- 청구기호
- 저자명
- 서명/저자
- studies of the biosynthesis of bacterial cell wall and antibiotic deoxysugar moieties: dtdp-rhamnose, cdp-ascarylose, and dtdp-mycaminose (yersinia pseudotuberculosis, streptomyces fradiae). - [microform]
- 발행사항
- 형태사항
- 285 p. : microfiches ; 11×15 cm.
- 총서명
- UMI Dissertation
- 주기사항
- Source: Dissertation Abstracts International, Volume: 58-02, Section: B, page: 0685.
- 학위논문주기
- thesis (ph.d.)-- - university of minnesota, 1997.
- 초록/해제
- 요약Deoxysugars are ubiquitous components of various natural products that aid or confer upon these molecules specific and often crucial biological properties. In this treatise, an effort to contribute to a better understanding of the enzymes involved in the intricate deoxysugar biosynthetic machinery invoked by bacteria is described. Three of the four enzymes scrutinized--dTDP-6-deoxy- scL-threo- scD-glycero-4-hexulose 3,5-epimerase (rfbC), dTDP-6-deoxy- scL-lyxo-4-hexulose 4-reductase (rfbD), and CDP-6-deoxy- scL-threo- scD-glycero-4-hexulose-3-dehydrase (E$\sb1$)--are key participants in the formation of deoxysugars that are integral components of the protective bacterial Gram-negative cell membrane. Cloning, overexpression, homogeneous purification, and characterization of the two rhamnose biosynthetic enzymes, the epimerase rfbC and the reductase rfbD, from Salmonella enterica LT2 were achieved. This development afforded a more detailed inspection of their respective catalytic mechanisms. Of note is the fortuitous discovery of an intriguing facet of rfbD NADH-dependent reduction--the absence of stereospecificity in hydride transfer when the alternate electron acceptor dichlorophenolindophenol (DCPIP) is used in lieu of the sugar substrate. Further studies on the third enzyme E$\sb1,$ a novel deoxygenating pyridoxamine phosphate (PMP)-dependent enzyme in the 3,6-dideoxyhexose (ascarylose) biosynthetic pathway of Yersinia pseudotuberculosis, was also carried out. A delineation of an interesting property--as surrogate cofactor-binder and catalytic-effector--of the proximal E$\sb1$ active site residue His-221, in relation to the catalytic and PMP-binding active site moiety His-220, via site directed mutagenesis and validation by enzyme assays was thus attained. Our initial foray into the biosynthesis of the deoxysugars contained in the macrolide antibiotic tylosin, of mycaminose in particular, resulted in the sequencing of the tylIBA gene cluster encoding for this aminodeoxysugar, sequence data analysis, and gene identification. Coupled with sequencing data from the tylLM region, the biogenesis of dTDP-mycaminose in Streptomyces fradiae is postulated to involve an amination step resulting from the cooperative efforts of tylB and tylX3 and possibly by tylX5. Possible parallels between this proposed amino group incorporation in mycaminose formation and those in p-aminobenzoic acid biosynthesis are cited. Finally, the amplification, cloning, overexpression, and initial characterization of the putative transaminase tylB are discussed, including various attempts at inclusion body solubilization and reconstitution.
- 복제주기
- Microfiche : UMI . microfiches;11×15 cm.
- 일반주제명
- 일반주제명
- 일반주제명
- 키워드
- 기타저자
- 기본자료저록
- Dissertation Abstracts International. 58-02B.
MARC
008970923s1997 us eng■001MOKWON00235190
■001AAV9721636
■00519981008111140
■008970923s1997 us eng
■035 ▼a(UnM)AAV9721636
■040 ▼aUnM▼cUnM▼dMOKWON
■090 ▼a540▼bQ3s
■1001 ▼aque, nanette loida sanidad.
■24510▼astudies of the biosynthesis of bacterial cell wall and antibiotic deoxysugar moieties: dtdp-rhamnose, cdp-ascarylose, and dtdp-mycaminose (yersinia pseudotuberculosis, streptomyces fradiae).▼h[microform]
■260 ▼aU.S.▼buniversity of minnesota▼c1997.
■300 ▼a285 p.▼bmicrofiches▼c11×15 cm.
■350 ▼a$50.6
■44000▼aUMI Dissertation
■500 ▼aSource: Dissertation Abstracts International, Volume: 58-02, Section: B, page: 0685.
■502 ▼athesis (ph.d.)--▼buniversity of minnesota▼d1997.
■520 ▼aDeoxysugars are ubiquitous components of various natural products that aid or confer upon these molecules specific and often crucial biological properties. In this treatise, an effort to contribute to a better understanding of the enzymes involved in the intricate deoxysugar biosynthetic machinery invoked by bacteria is described. Three of the four enzymes scrutinized--dTDP-6-deoxy- scL-threo- scD-glycero-4-hexulose 3,5-epimerase (rfbC), dTDP-6-deoxy- scL-lyxo-4-hexulose 4-reductase (rfbD), and CDP-6-deoxy- scL-threo- scD-glycero-4-hexulose-3-dehydrase (E$\sb1$)--are key participants in the formation of deoxysugars that are integral components of the protective bacterial Gram-negative cell membrane. Cloning, overexpression, homogeneous purification, and characterization of the two rhamnose biosynthetic enzymes, the epimerase rfbC and the reductase rfbD, from Salmonella enterica LT2 were achieved. This development afforded a more detailed inspection of their respective catalytic mechanisms. Of note is the fortuitous discovery of an intriguing facet of rfbD NADH-dependent reduction--the absence of stereospecificity in hydride transfer when the alternate electron acceptor dichlorophenolindophenol (DCPIP) is used in lieu of the sugar substrate. Further studies on the third enzyme E$\sb1,$ a novel deoxygenating pyridoxamine phosphate (PMP)-dependent enzyme in the 3,6-dideoxyhexose (ascarylose) biosynthetic pathway of Yersinia pseudotuberculosis, was also carried out. A delineation of an interesting property--as surrogate cofactor-binder and catalytic-effector--of the proximal E$\sb1$ active site residue His-221, in relation to the catalytic and PMP-binding active site moiety His-220, via site directed mutagenesis and validation by enzyme assays was thus attained. Our initial foray into the biosynthesis of the deoxysugars contained in the macrolide antibiotic tylosin, of mycaminose in particular, resulted in the sequencing of the tylIBA gene cluster encoding for this aminodeoxysugar, sequence data analysis, and gene identification. Coupled with sequencing data from the tylLM region, the biogenesis of dTDP-mycaminose in Streptomyces fradiae is postulated to involve an amination step resulting from the cooperative efforts of tylB and tylX3 and possibly by tylX5. Possible parallels between this proposed amino group incorporation in mycaminose formation and those in p-aminobenzoic acid biosynthesis are cited. Finally, the amplification, cloning, overexpression, and initial characterization of the putative transaminase tylB are discussed, including various attempts at inclusion body solubilization and reconstitution.
■533 ▼aMicrofiche▼cUMI▼emicrofiches;11×15 cm.
■590 ▼aSchool code: 0130.
■650 4▼aChemistry, Biochemistry
■650 4▼aChemistry, Organic
■650 4▼aBiology, Microbiology
■653 ▼astudies▼aof▼athe▼abiosynthesis▼aof▼abacterial▼acell▼awall▼aand▼aantibiotic▼adeoxysugar▼amoieties▼a▼adtdp-rhamnose▼acdp-ascarylose▼aand▼adtdp-mycaminose▼a▼a(yersinia▼apseudotuberculosis▼astreptomyces▼afradiae).
■690 ▼a0487
■690 ▼a0490
■690 ▼a0410
■71020▼auniversity of minnesota.
■7730 ▼tDissertation Abstracts International▼g58-02B.
■790 ▼a0130
■791 ▼aPH.D.
■792 ▼a1997
