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Potassium-activated GTPase reaction in the G Protein-coupled ferrous iron transporter B.

ABSTRACT: FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe(2+)). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe(2+) membrane transport mechanism. Here, we present crystal structures of the soluble domain of FeoB from Streptococcus thermophilus in complex with GDP and with the GTP analogue derivative 2'-(or -3')-O-(N-methylanthraniloyl)-beta,gamma-imidoguanosine 5'-triphosphate (mant-GMPPNP). Unlike recent structures of the G protein domain, the mant-GMPPNP-bound structure shows clearly resolved, active conformations of the critical Switch motifs. Importantly, biochemical analyses demonstrate that the GTPase activity of FeoB is activated by K(+), which leads to a 20-fold acceleration in its hydrolysis rate. Analysis of the structure identified a conserved asparagine residue likely to be involved in K(+) coordination, and mutation of this residue abolished K(+)-dependent activation. We suggest that this, together with a second asparagine residue that we show is critical for the structure of the Switch I loop, allows the prediction of K(+)-dependent activation in G proteins. In addition, the accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter.

SUBMITTER: Ash MR

PROVIDER: S-EPMC2863241 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Potassium-activated GTPase reaction in the G Protein-coupled ferrous iron transporter B.

Ash Miriam-Rose MR Guilfoyle Amy A Clarke Ronald J RJ Guss J Mitchell JM Maher Megan J MJ Jormakka Mika M

The Journal of biological chemistry 20100310 19

FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe(2+)). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe(2+) membrane transport mechanism. Here, we present crystal structures of the soluble domain of FeoB from Strepto ...[more]

PMID: 20220129

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Project description:The AdhR regulatory protein is an activator of σ54-dependent transcription of adhA1 and adhA2 genes, which are required for alcohol synthesis in Clostridium beijerinckii Here, we identified the signal perceived by AdhR and determined the regulatory mechanism of AdhR activity. By assaying the activity of AdhR in N-terminally truncated forms, a negative control mechanism of AdhR activity was identified in which the central AAA+ domain is subject to repression by the N-terminal GAF and PAS domains. Binding of Fe2+ to the GAF domain was found to relieve intramolecular repression and stimulate the ATPase activity of AdhR, allowing the AdhR to activate transcription. This control mechanism enables AdhR to regulate transcription of adhA1 and adhA2 in response to cellular redox status. The mutants deficient in AdhR or σ54 showed large shifts in intracellular redox state indicated by the NADH/NAD+ ratio under conditions of increased electron availability or oxidative stress. We demonstrated that the Fe2+-activated transcriptional regulator AdhR and σ54 control alcohol synthesis to maintain redox homeostasis in clostridial cells. Expression of N-terminally truncated forms of AdhR resulted in improved solvent production by C. beijerinckiiIMPORTANCE Solventogenic clostridia are anaerobic bacteria that can produce butanol, ethanol, and acetone, which can be used as biofuels or building block chemicals. Here, we show that AdhR, a σ54-dependent transcriptional activator, senses the intracellular redox status and controls alcohol synthesis in Clostridium beijerinckii AdhR provides a new example of a GAF domain coordinating a mononuclear non-heme iron to sense and transduce the redox signal. Our study reveals a previously unrecognized functional role of σ54 in control of cellular redox balance and provides new insights into redox signaling and regulation in clostridia. Our results reveal AdhR as a novel engineering target for improving solvent production by C. beijerinckii and other solventogenic clostridia.

Dataset Information

Potassium-activated GTPase reaction in the G Protein-coupled ferrous iron transporter B.

Publications

Potassium-activated GTPase reaction in the G Protein-coupled ferrous iron transporter B.

Similar Datasets

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