Investigations in Enzyme Stereospecificity: I. The Stereochemistry of Cis-Double Bond Formation in Dolichol and Natural Rubber Biosynthesis. II. The Stereochemistry of Carbon-Sulfur 4ond Reduction by Methyl Coenzyme Reductase
Schlicher, Martha Ann
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https://hdl.handle.net/2142/70432
Description
Title
Investigations in Enzyme Stereospecificity: I. The Stereochemistry of Cis-Double Bond Formation in Dolichol and Natural Rubber Biosynthesis. II. The Stereochemistry of Carbon-Sulfur 4ond Reduction by Methyl Coenzyme Reductase
Author(s)
Schlicher, Martha Ann
Issue Date
1988
Doctoral Committee Chair(s)
Coates, Robert M.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Organic
Abstract
Dolichol, a poly-cis polyprenol, is biosynthesized from isopentenyl pyrophosphate, IPP, and farnesyl pyrophosphate, FPP, as a branch from the pathway to squalene and cholesterol. Natural rubber, also a poly-cis polyprenol, is biosynthesized from Hevea latex in the same manner. The stereochemical details of the condensation of IPP with the pro-chiral center of the developing dolichol and rubber chains are important in both analysis of the spatial arrangement of the enzyme-substrate complex and in the design of inhibitors or regulators for the pathway. Tritium labeled dehydrodolichol and natural rubber were biosynthesized from both (1S)- (1-$\sp3$H$\sb1$) IPP and (E)- (4-$\sp3$H$\sb1$) IPP by Dr. Hiroshi Sagami at Tohoku University in Sendai, Japan. The configuration of the tritium label located at the newly generated allylic position in the biosynthesized rubber and dehydrodolichol chains was determined in three of the four products. Elucidation of the stereochemistry of the products was accomplished through reductive ozonolysis to 1,4-pentanediol and 2-methyl 2,5-hexanediol followed by resolution of the diol enantiomers and stereospecific degradation. The key step in both degradations was a selenoxide syn elimination to form a mixture of both E and Z alkenes.
In agreement with Cornforth's and Popjak's results studying trans-polyprenol biosynthesis, the carbinyl carbons of the cis-polyprenols, dolichol and rubber, undergo an inversion of configuration during alkylation with IPP. Evidence is also provided for an overall syn facial addition-elimination in the condensation to form natural rubber with alkylation occurring from the same face as previously established hydrogen elimination. Results were inconclusive in determining the faciality of attack in dolichol biosynthesis.
Chiral (R)- (1-$\sp2$H$\sb1$,1-$\sp3$H$\sb1$) ethyl coenzyme was prepared for the elucidation of the stereochemistry of the reduction of the C-S bond catalyzed by methyl CoM reductase from Methanobacterium thermoautotrophium. Stereospecific reduction of (1-$\sp3$H$\sb1$) acetaldehyde with YAD and NADD provided (1S)- (1-$\sp3$H$\sb1$,1-$\sp2$H$\sb1$) ethanol which was isolated as the trisylate before conversion to the coenzyme. Analysis of the stereochemistry of the doubly labeled ethane biosynthesized from EtCoM with methyl CoM reductase will be accomplished by conversion to chiral acetate and analysis by the chiral methyl assay procedure.
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