Differential effects of a molybdopterin synthase sulfurylase (moeB) mutation on Escherichia coli molybdoenzyme maturation.
We have generated a chromosomal mutant of moeB (moeBA228T) that demonstrates limited molybdenum cofactor (molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD)) availability in Escherichia coli and have characterized its effect on the maturation and physiological function of two well-characterized respiratory molybdoenzymes: the membrane-bound dimethylsulfoxide (DMSO) reductase (DmsABC) and the membrane-bound nitrate reductase A (NarGHI). In the moeBA228T mutant strain, E. coli F36, anaerobic respiratory growth is possible on nitrate but not on DMSO, indicating that cofactor insertion occurs into NarGHI but not into DmsABC. Fluorescence analyses of cofactor availability indicate little detectable cofactor in the moeBA228T mutant compared with the wild-type, suggesting that NarGHI is able to scavenge limiting cofactor, whereas DmsABC is not. MoeB functions to sulfurylate MoaD, and in the structure of the MoeB-MoaD complex, Ala-228 is located in the interface region between the two proteins. This suggests that the moeBA228T mutation disrupts the interaction between MoeB and MoaD. In the case of DmsABC, despite the absence of cofactor, the twin-arginine signal sequence of DmsA is cleaved in the moeBA228T mutant, indicating that maturation of the holoenzyme is not cofactor-insertion dependent.
Sambasivarao
Damaraju
D
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Turner
Raymond J
RJ
Bilous
Peter T
PT
Rothery
Richard A
RA
Shaw
Gillian
G
Weiner
Joel H
JH
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D015153:Blotting, Western; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D009565:Nitrate Reductases / Q000378:metabolism*; D009713:Nucleotidyltransferases; D010088:Oxidoreductases / Q000378:metabolism*; D017354:Point Mutation; D011499:Protein Processing, Post-Translational / Q000235:genetics; D013050:Spectrometry, Fluorescence; D017973:Tungsten Compounds / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D017973:Tungsten Compounds; C403342:moeB protein, Bacteria; D010088:Oxidoreductases; D009565:Nitrate Reductases; C045664:dimethyl sulfoxide reductase; C499585:MoeB protein, E coli; D009713:Nucleotidyltransferases; C045951:tungstate