The Paradoxical Effect of Paraquat on Leucyl-tRNA Levels in &amp;lt;i&amp;gt;E. coli&amp;lt;/i&amp;gt; Provides New Insights for Amino Acid Therapy in Humans

Babu V Bassa,Olen R Brown

doi:10.4236/cellbio.2018.74005

Abstract

Dihydroxyacid dehydratase (DHAD), the rate-limiting enzyme in the synthesis of branched-chain (BCA) amino acids in bacteria and plants, is sensitive to oxyradical toxicity. Oxidant stress reversibly inactivates DHAD and causes starvation for BCA and reversible cessation of growth in Escherichia coli [1][2]. To better understand the underlying toxicity mechanisms, we have determined the cellular concentrations of charged-tRNAs for BCA, in E. coli treated with the redox-active chemical, paraquat. Contrary to expectation, in the paraquat-treated cells, the concentration of only charged leucyl-tRNA decreased dramatically; whereas, the concentrations of the other BCAs (valine and isoleucine) increased. This paradoxical result, the “paraquat effect” can be best explained if leucine is the most abundant amino acid in the E. coli proteins and therefore the rate-limiting building block in their synthesis. Based on this assumption, we investigated the concentration of free amino acids in E. coli and their relative abundances in E. coli proteins. Protein amino acid frequencies were determined by analyzing one-hundred gene bank protein sequences with software developed as described in Methods. Leucine is the most abundant amino acid in the E. coli proteins (10%) and consequently, the cellular free leucine concentration is smaller and the native charged-leucyl-tRNA levels are much higher than those of valine and isoleucine. This has relevance to humans because: leucine-deprivation was shown to be beneficial in tumor suppression [3], and leucine-supplementation was beneficial in the recovery from exercise-induced muscle loss [4][5], and leucine also occurs at a higher frequency in almost all human proteins. In three human protein categories, we examined it ranged from 9% to 17%. This predominance of leucine in proteins would make cells vulnerable to impairment of the leucine pools and could explain our results in E. coli and some of the biological effects of free leucine in humans.

Highlights

Exposure of E. coli K-12 to the oxidative stress of aerobic paraquat, hyperbaric oxygen, or other oxidants, initiates a sequence of events leading to the induction of genetic stringency, a process discovered by Caschel [6] [7]
We demonstrated through our earlier work [9] [10] that paraquat causes oxidant stress similar to hyperbaric oxygen and other redox-cycling compounds and reversibly inactivates Dihydroxyacid dehydratase (DHAD) in E. coli K-12
In the present project we have directly demonstrated the effect of paraquat on the cellular content of charged branched- chain amino acid tRNAs in the E. coli K-12 strain

Summary

Introduction

Exposure of E. coli K-12 to the oxidative stress of aerobic paraquat, hyperbaric oxygen, or other oxidants, initiates a sequence of events leading to the induction of genetic stringency, a process discovered by Caschel [6] [7]. Amino acid pools are depleted and unloaded tRNAs are the “trigger” that induce ribosomes of stringent type cells (which contain the stringency protein) to synthesize guanosine tetra phosphate (ppGpp) [6] [8], which in turn binds to RNA polymerase [6] [8] and inhibits the synthesis of RNA. Oxidant stress inactivates the [4Fe-4S] cluster of DHAD(2) and thereby causes stringency, in minimal medium, by the deprivation of BCA. We demonstrated through our earlier work [9] [10] that paraquat causes oxidant stress similar to hyperbaric oxygen and other redox-cycling compounds and reversibly inactivates DHAD in E. coli K-12. Paraquat toxicity in E. coli K-12 is at least partly mediated, through its effects on DHAD, because supplementation of branched-chain amino acids mitigates the toxic effects of paraquat [10] via this mechanism

Methods

Results

Conclusion