The present studies have shown that GSH metabolism arose in the purple bacteria and cyanobacteria where it functions to protect against oxygen toxicity. Evidence was obtained indicating that GSH metabolism was incorporated into eucaryotes via the endosymbiosis giving rise to mitochrondria and chloroplasts. Aerobic bacteria lacking GSH utilize other thiols for apparently similar functions, the thiol being coenzyme A in Gram positive bacteria and chi-glutamylcysteine in the halobacteria. The thiol biochemistry of prokaryotes is thus seen to be much more highly diversified than that of eucaryotes and much remains to be learned about this subject

Fahey, Robert C.

English

NASA Technical Reports Server

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FINAL TECHNICAL REPORT 
NASA GRANT NAGW-342 i 
THIOL BIOCHEMISTRY OF PROKARYOTES 
Pr inc ipa l  Investigator:  Robert C .  Fahey J 
Department of Chemistry D-006 
University of California,  San Diego 
La Jolla CA 92093 
(619) 534-2163, 534-4821 
Grantee Ins t i tu t ion :  University of California,  San Diego 
Period Covered: June 7 ,  1982 through December 31, 1986 
(bASA-CR-780257)  THICL B I C C E E @ I S I B P  OP B E ? - 1  1 5 7 9  
E S G K A B Y C I E S  E i n a l  Technical  E e p r t ,  7 Jun, 
IS82 - 3 1  Dec. 19€6 ( C a l i f c r c i a  Oniv., San 
Liego, La J o l l a . )  5 p C S C L  06P Unclas 
G3/52 43816 
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https://ntrs.nasa.gov/search.jsp?R=19870009546 2020-03-20T11:31:37+00:00Z
BRIEF SUMMARY OF PROJECT 
Low molecular weight thiols play a key role in cellular defenses against 
oxygen toxicity. 
shown that it is absent in many prokaryotes. 
have existed in prokaryotes prior to the evolution of cyanobacteria and 
accumulation of oxygen in the atmosphere, it might be expected that 
evolutionarily distinct groups of bacteria evolved mechanisms of protection 
against oxygen toxicity based upon different low molecular weight thiols. The 
long term goal of this research is to delineate the differences in the thiol 
biochemistry of prokaryotes and to understand how these differences evolved in 
response to the shift from anaerobic to aerobic metabolism. 
this proposed research are: (1) to expand the methods of thiol analysis 
developed in our laboratory to measure all known biological thiols and to 
include analyses for thioesters, disufides , and dithiol proteins (thioredoxin) ; 
(2) to apply these methods to establish the main low molecular weight thiols 
produced by all major groups of prokaryotes; (3) to establish the structures of 
any novel thiols which appear to have wide distribution; (4) to elaborate the 
occurence of key enzymes, especially disulfide reductases, involved in the 
metabolism of these thiols. 
Glutathione is the thiol used in higher organisms but we have 
Since substantial diversity must 
Specific aims of 
With respect to the first specific aim, the methodology based upon 
fluorescent labeling with monobromobimane for analysis of thiols and thiol 
derivatives has been greatly extended and now includes ion pairig HPLC 
techniques which permit analysis of low molecular weight thiols that are highly 
anionic in character, such as coenzyme A, as well as cationic thiols (Pl,P7). 
Methods for specific analysis of the thiol components of thioesters and 
disulfides proved more difficult to develop than expected owing to the fact that 
conventional methods used to cleave disulfides also cleave thioesters but 
selective cleavage based upon hydroxylamine led to a successful method (P4). 
An analysis for thioredoxin was also developed based upon fluorescent labeling 
of the two cysteine residues and HPK analysis after a heat treatment and 
desalting procedure (P5). To facilitate isolation and identification of thiols 
of unknown or uncertain structure from biological systems a rapid and efficient 
method for purification of thiols from biological extracts was developed based 
upon thiol affinity chromatography and preparative HPLC (P8). 
In accomplishing specific aims (2) and (3) we have shown that glutathione 
Since the purple bacteria are 
is absent in all strictly anaerobic bacteria studied and is produced only by the 
purple bacteria and the cyanobacteria (Pl, ~6). 
the group most closely related to mitochondria and the cyanobacteria are related 
to chloroplasts, this suggests that incorporation of glutathione metabolism into 
eucaryotes could have occurred during the endosymbiosis giving rise to 
mitochondria and chloroplasts. This hypothesis is supported by the finding that 
Entamoeba histolytica, a eucaryote lacking both mitochondria and chloroplasts, 
does not produce glutathione or the enzymes of glutathione metabolism (P2). To 
further understand mechanisms of protection against oxygen toxicity it was 
important to ascertain what thiols are used by aerobic bacteria which lack GSH. 
The Gram positive bacteria and the radiobacteria were found to produee coenzyme 
A in unusually large amounts (Pl) whereas, among the archaebacteria, the 
halobacteria were shown to produce half of the glutathione molecule, 
1-glutangrlcysteine, as the major intracellular thiol (P3). 
unidentified thiols were also detected, but few occurred at high levels and had 
wide distribution. 
A wide variety of 
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The pursuit of objective ( 4 )  was confined primarily to studies of the 
disulfide reductases responsible for maintaining coenzyme A in a reduced state 
in Gram positive bacteria and for reducing 1-glutamylcysteine disulfide in 
halobacteria. At least two coenzyme A disulfide reductases have been identified 
in Bacillus cereus (Al,A5) and a 1-glutaqylcysteine disulfide reductase has 
been detected in halobacteria (P3). The latter has been purified to homogeneity - 
and is presently being characterized (A6, A. R. Sundquist-and R. C. Fahey, 
unpublished); a fuJ-1 report on this enzyme will be submitted in mid-1987. 
In conclusion, the present studies have shown that GSH metabolism arose in 
the purple bacteria and cyanobacteria where it functions to protect against 
oxygen toxicity. 
incorporated into eucaryotes via the endosymbiosis giving rise to mitochondria 
and chloroplasts. 
apparently similar functions, the thiol being coenzyme A in Gram positive 
bacteria and 1-glutamylcysteine in the halobacteria. 
prokaryotes is thus seen to be much more highly diversified than that of 
eucaryotes and much remains to be learned about this subject. 
Evidence was obtained indicating that GSH metabolism was 
Aerobic bacteria lacking GSH utilize other thiols for 
The thiol biochemistry of 
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ABSTRACTS AND PAPERS PRESENTED AT MEETINGS 
Al. Isolation and Initial Characterization of a Novel Disulfide Reductase from 
Bacillus cereus. R. C. Fahey and M. Limova, Fed. Proc. 42, 1964 (1983). 
A2. Low Molecular Weight Thiols in Halobacteria. G. L. Newton, B. J. Javor, 
and R. C. Fahey, West Coast Bacterial Physiologists Annual Asilomar 
Conference, December 15-17, 1984. 
A3. Determination of the Thiol Components of Biologically Important Thioesters 
and Disulfides. S. S. Fenton and R. C. Fahey, Fed. Proc. 44, 1211 (1985). 
Ah. Fluorescent Labeling of E. coli Thioredoxin by Monobromobimane. P. C. 
Chinn, V. Pigiet, and R. C. Fahey, Fed. Proc. 44, 1620 (1985). 
A5. Partial Purification and Characterization of an NAD( P)H Dependent Coenzyme 
A Disulfide Reductase from Bacillus cereus. T. D. Chicken and R. C. Fahey, 
West Coast Bacterial Physiolgists Annual Asilomar Conference, December 
13-15, 1985. 
A6. Partial Purification and Characterization of a Bis-1-Glutaqylcystine 
Reductase from Halobacterium halobium. A. R. Sundquist and R. C. Fahey, 
West Coast Bacterial Physiolgists Annual Asilomar Conference, December 
13-15, 1985. 
A7. Evolution of Thiol Protective Systems in Prokaryotes. R. C. Fahey and G. 
L. 
Evolution of Life, Moffett Field, California, July 23-26, 1985. 
Newton, Second Symposium on Chemical Evolution and the Origin and 
A8. Evolution of Glutathione Metabolism in Phototrophic Microorganisms. R. C. 
Fahey, R. M. Buschbacher, and G. L. Newton, Fifth ISSOL Meeting and Eighth 
International Conference on the Origin of Life, Berekely, July 21-25, 1986. 
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PUBLICATIONS 
P1. Occurrence of Low Molecular Weight Thiols in Biological Systems. R. C. 
Fahey and G. L. Newton, in "Functions of Glutathione - Biochemical, 
Physiological, and Toxicological Aspects", A. Larsson, et al, eds. , Raven 
Press, New York, pp. 252-260 (1983). 
P2. Entamoeba histolytica: A Eukaryote Without Glutathione Metabolism. R. C. 
Fahey, G. L. Newton, B. Arrick, T. Overdank-Bogart, S. B. Aley, Science, 
224, 70-72 (1984). 
P3. 1-Glutamylcysteine and Thiosulfate Are the Major Low-Molecular-Weight 
Thiols in Halobacteria. G. L. Newton and B. Javor, J. Bacteriol. 161, 
438-441 (1985 1. 
P4. Analysis of Biological Thiols: Determination of Thiol Components of 
Disulfides and Thioesters. S .  S. Fenton and R. C. Fahey, Anal. Biochem. 
154, 34-42 (1986). 
P5. Determination of Thiol Proteins Using Monobromobimane Labeling and 
High-Performance Liquid Chromatographic Analysis: 
Escherichia coli Thioredoxin. P. C. Chinn, V. Pigiet, and R. C. Fahey, 
Anal. Biochem. 159, 143-149 (1986). 
Application to 
~ 6 .  The Evolution of Glutathione Metabolism in Phototrophic Microorganisms. R. 
C. Fahey, R. M. Buschbacher, and G. L. Newton, J. Mol. Evol., in press 
( 1987 
P7. Determination of Low Molecular Weight Thiols Using Monobromobimane 
Fluorescent Labeling and High-Performance Liquid Chromatography. R. C. 
Fahey and G. L. Newton, Methods Enzymol. , in press (1987). 
P8. Purification of Thiols from Biological Samples. G. L. Newton and R. C. 
Fahey, Methods Enzymol. , in press (1987). 
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Thiol biochemistry of prokaryotes

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