The principal aim of this investigation is to understand the regulation of expression of the genes and enzymes involved in the vitamin K biosynthetic pathway. Vitamin K is an important fat soluble vitamin that is required for the well being of humans and animals. Vitamin K responsive hypoprothrombia is an important clinical problem under a number of conditions. The bacterial flora of the human intestine contribute from 50-100% of the human vitamin K requirement. Vitamin K is derived from the """"""""shikimate pathway"""""""" and it is formed from isochorismate and 2-ketoglutarate. The intermediates that have been identified are 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC), o-succinylbenzoate (OSB), a CoA ester of OSB (OSB-CoA), 1,4- dihydroxy-2-naphthoate (DHNA), and desmethylmenaquinone (DMK). The menD, C, E, and B genes located at 48.5 min of the chromosome have been cloned and sequenced. The sequencing of the menA gene at 89 min is in progress and we will identify and clone the gene for the conversion of DMK---> MK. To overcome the difficulty of working with small quantities of enzymes and intermediates, we will use operon and protein fusions to study regulation and we will introduce the genes into overexpression vectors to overproduce, purify and study the enzymes and intermediates of the pathway.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050262-02
Application #
2187964
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1994-07-01
Project End
1998-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
2
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Northern Illinois University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
De Kalb
State
IL
Country
United States
Zip Code
60115
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Palmer, D R; Garrett, J B; Sharma, V et al. (1999) Unexpected divergence of enzyme function and sequence: ""N-acylamino acid racemase"" is o-succinylbenzoate synthase. Biochemistry 38:4252-8
Suvarna, K; Stevenson, D; Meganathan, R et al. (1998) Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menA gene from Escherichia coli. J Bacteriol 180:2782-7
Bhattacharyya, D K; Kwon, O; Meganathan, R (1997) Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase. J Bacteriol 179:6061-5
Daruwala, R; Bhattacharyya, D K; Kwon, O et al. (1997) Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli. J Bacteriol 179:3133-8
Kwon, O; Hudspeth, M E; Meganathan, R (1996) Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis. J Bacteriol 178:3252-9
Daruwala, R; Kwon, O; Meganathan, R et al. (1996) A new isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis encoded by the menF gene. FEMS Microbiol Lett 140:159-63
Kwon, O; Bhattacharyya, D K; Meganathan, R (1996) Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and properties of o-succinylbenzoyl-coenzyme A synthetase from Escherichia coli. J Bacteriol 178:6778-81
Sharma, V; Hudspeth, M E; Meganathan, R (1996) Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menE gene from Escherichia coli. Gene 168:43-8