Functional and structural analysis of members of the TorD family, a large chaperone family dedicated to molybdoproteins

The trimethylamine N-oxide (TMAO) reductase TorA, a DMSO reductase family member, is a periplasmic molybdoenzyme of Escherichia coli. The cytoplasmic protein TorD acts as a chaperone for TorA, allowing the efficient insertion of the molybdenum cofactor into the apoform of the enzyme prior to its secretion. This paper demonstrates that TorD is a member of a large family of prokaryotic proteins that are structurally related. Moreover, their genes generally belong to operons also encoding molybdoenzymes of the DMSO reductase family. Both the TorD and the DMSO reductase families present a similar phylogenetic organization, suggesting a co-evolution of these two families of proteins. This hypothesis is also supported by the fact that the TorD and DmsD chaperones cannot replace each other and thus appear dedicated to specific molybdopartners. Interestingly, it was found that the positive effect of TorD on TorA maturation, and the partial inhibitory effect of DmsD and homologues, are independent of the TorA signal sequence.

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Use of Thioredoxin as a Reporter To Identify a Subset of Escherichia coli Signal Sequences That Promote Signal Recognition Particle-Dependent Translocation

Journal of Bacteriology ◽

10.1128/jb.187.9.2983-2991.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 2983-2991 ◽

Cited By ~ 95

Author(s):

Damon Huber ◽

Dana Boyd ◽

Yu Xia ◽

Michael H. Olma ◽

Mark Gerstein ◽

...

Keyword(s):

Escherichia Coli ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Cytoplasmic Protein ◽

Signal Sequences ◽

Simple Measure ◽

Cotranslational Translocation ◽

Dependent Signal ◽

Thioredoxin 1

ABSTRACT We have previously reported that the DsbA signal sequence promotes efficient, cotranslational translocation of the cytoplasmic protein thioredoxin-1 via the bacterial signal recognition particle (SRP) pathway. However, two commonly used signal sequences, those of PhoA and MalE, which promote export by a posttranslational mechanism, do not export thioredoxin. We proposed that this difference in efficiency of export was due to the rapid folding of thioredoxin in the cytoplasm; cotranslational export by the DsbA signal sequence avoids the problem of cytoplasmic folding (C. F. Schierle, M. Berkmen, D. Huber, C. Kumamoto, D. Boyd, and J. Beckwith, J. Bacteriol. 185 :5706-5713, 2003). Here, we use thioredoxin as a reporter to distinguish SRP-dependent from non-SRP-dependent cleavable signal sequences. We screened signal sequences exhibiting a range of hydrophobicity values based on a method that estimates hydrophobicity. Successive iterations of screening and refining the method defined a threshold hydrophobicity required for SRP recognition. While all of the SRP-dependent signal sequences identified were above this threshold, there were also a few signal sequences above the threshold that did not utilize the SRP pathway. These results suggest that a simple measure of the hydrophobicity of a signal sequence is an important but not a sufficient indicator for SRP recognition. In addition, by fusing a number of both classes of signal sequences to DsbA, we found that DsbA utilizes an SRP-dependent signal sequence to achieve efficient export to the periplasm. Our results suggest that those proteins found to be exported by SRP-dependent signal sequences may require this mode of export because of their tendency to fold rapidly in the cytoplasm.

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Differential effects of a molybdopterin synthase sulfurylase (moeB) mutation on Escherichia coli molybdoenzyme maturation

Biochemistry and Cell Biology ◽

10.1139/o02-131 ◽

2002 ◽

Vol 80 (4) ◽

pp. 435-443 ◽

Cited By ~ 10

Author(s):

Damaraju Sambasivarao ◽

Raymond J Turner ◽

Peter T Bilous ◽

Richard A Rothery ◽

Gillian Shaw ◽

...

Keyword(s):

Escherichia Coli ◽

Nitrate Reductase ◽

Mutant Strain ◽

Physiological Function ◽

Signal Sequence ◽

Molybdenum Cofactor ◽

Wild Type ◽

Dmso Reductase ◽

E Coli ◽

Membrane Bound

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 MoeBMoaD 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.Key words: mdybdenum cofactor, DMSO reductase, nitrate reductase.

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Enzymatic and physiological properties of the tungsten-substituted molybdenum TMAO reductase from Escherichia coli

Molecular Microbiology ◽

10.1046/j.1365-2958.1999.01340.x ◽

1999 ◽

Vol 32 (1) ◽

pp. 159-168 ◽

Cited By ~ 59

Author(s):

Jean Buc ◽

Claire-Lise Santini ◽

Roger Giordani ◽

Mirjam Czjzek ◽

Long-Fei Wu ◽

...

Keyword(s):

Escherichia Coli ◽

Physiological Properties ◽

Tmao Reductase

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Intragenic reversion mutations that improve export of maltose-binding protein in Escherichia coli malE signal sequence mutants.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)35795-2 ◽

1986 ◽

Vol 261 (7) ◽

pp. 3389-3395

Author(s):

J P Ryan ◽

M C Duncan ◽

V A Bankaitis ◽

P J Bassford

Keyword(s):

Escherichia Coli ◽

Signal Sequence ◽

Binding Protein ◽

Maltose Binding Protein

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Characterization and in Vivo Cloning of prlC, a Suppressor of Signal Sequence Mutations in Escherichia coli K12

Genetics ◽

10.1093/genetics/116.4.513 ◽

1987 ◽

Vol 116 (4) ◽

pp. 513-521

Author(s):

Nancy J Trun ◽

Thomas J Silhavy

Keyword(s):

Escherichia Coli ◽

Genetic Mapping ◽

Signal Sequence ◽

Illegitimate Recombination ◽

Mutant Phenotype ◽

E Coli ◽

Physical Location ◽

Sequence Deletion ◽

New Alleles

ABSTRACT The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamBsignal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using λplacMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

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Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

Scientific Reports ◽

10.1038/s41598-021-95123-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Charles R. Midgett ◽

Kacey Marie Talbot ◽

Jessica L. Day ◽

George P. Munson ◽

F. Jon Kull

Keyword(s):

Escherichia Coli ◽

Small Molecules ◽

Family Member ◽

Shigella Flexneri ◽

Decanoic Acid ◽

Enterotoxigenic Escherichia Coli ◽

Gram Negative Bacteria ◽

Enteric Infections ◽

First Time ◽

Arac Family

AbstractEnteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

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