Dihydroxy-acid dehydratase, a [4Fe-4S] cluster-containing enzyme in Escherichia coli: effects of intracellular superoxide dismutase on its inactivation by oxidant stress.
Dihydroxy-acid dehydratase (DHAD) has a [4Fe-4S] cluster and is reported to be facilely inactivated by oxidant stress. To directly assess the biological effects in vivo of superoxide dismutase (SOD) on the oxidant sensitivity of DHAD, we used an Escherichia coli K-12 parent strain (CGSC5073) and derived strains OB 1, OB 2, and OB 3 that lacked one of or both FeSOD and MnSOD. In the K-12 parent strain half the cellular DHAD activity was lost in 15 min at 0.8 atm oxygen, less than 10 microM aerobic nitrofurantoin, or about 5 microM aerobic paraquat (PQ) and in about 1 min at 10 microM aerobic PQ. Oxygen and metabolism were required for PQ to inactivate DHAD in cells; adding dithiothreitol to cell-free extracts did not restore DHAD activity. The Km was not appreciably changed for DHAD that was 50 and 70% inactivated in cells, respectively, by hyperbaric oxygen (HBO) and PQ, compared to cells in exponential, aerobic growth. Thus, active site oxidative impairment of individual enzyme molecules apparently was all-or-none. DHAD activity was greatly decreased when measured in extracts made from strains that lacked both SODs unless SOD was added to cell suspensions before extracts were made. DHAD was more sensitive in strains lacking both SODs than in the parent strain to inactivation by aerobic PQ and HBO. Anaerobic (compared to aerobic) growth increased DHAD specific activity by 20% or less in the parent strain and in strains OB 1 and OB 2 (lacking MnSOD and FeSOD, respectively); however, in strain OB 3 (lacking both SODs) DHAD was increased 60%. DHAD was partially inactivated by the oxidant stress of aerobic growth, but remained in a form detectable by DHAD antibody, and the ratio of active to inactive DHAD decreased greatly in cells lacking SOD. Thus, SOD helped maintain DHAD as an active holoenzyme and benefitted cells growing aerobically or when exposed to low levels of PQ.
Brown
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OR
John M. Dalton Cardiovascular Research Center, University of Missouri, Columbia 65211, USA.
Smyk-Randall
E
E
Draczynska-Lusiak
B
B
Fee
J A
JA
D002374:Catalase / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D007424:Intracellular Fluid / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D009582:Nitrofurantoin / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010269:Paraquat / Q000494:pharmacology; D013455:Sulfur / Q000737:chemistry; D013482:Superoxide Dismutase / Q000378:metabolism*
D013455:Sulfur; D009582:Nitrofurantoin; D007501:Iron; D002374:Catalase; D013482:Superoxide Dismutase; D006836:Hydro-Lyases; C020052:dihydroxyacid dehydratase; D010269:Paraquat; D004229:Dithiothreitol