L‐Serine and L‐threonine dehydratase from <i>Clostridium propionicum</i> Two enzymes with different prosthetic groups

Hofmeister, Antje; Grabowski, R.; Linder, Dietmar

doi:10.1111/j.1432-1033.1993.tb18040.x

Cited by 27 publications

(23 citation statements)

References 33 publications

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“…When purified aerobically, the enzyme is inactive until incubated with iron and DTT under aerobic conditions. These characteristics make it very similar to the LSDs from Clostridium acidiurici (17), Peptostreptococcus asaccharolyticus (18), and Clostridium propionicum (19), which are all activated in the same manner. Electron paramagnetic resonance (EPR) spectroscopic studies on the P. asaccharolyticus enzyme indicate the presence of [3Fe-4S] ϩ clusters (20).…”

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confidence: 74%

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Escherichia coli L-Serine Deaminase Requires a [4Fe-4S] Cluster in Catalysis

Cicchillo¹,

Baker²,

Schnitzer³

et al. 2004

Journal of Biological Chemistry

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L-Serine deaminases catalyze the deamination of Lserine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5 -phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5 -phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 ؎ 0.46 eq of iron and 0.670 ؎ 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying gʈ ‫؍‬ 2.03 and g Ќ ‫؍‬ 1.93. Mö ssbauer spectra of the 57 Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S] 2؉ clusters, as evidenced by the typical Mö ssbauer parameters-isomer shift, ␦ ‫؍‬ 0.47 mm/s, quadrupole splitting of ⌬E Q ‫؍‬ 1.14 mm/s, and a diamagnetic (S ‫؍‬ 0) ground state. Treatment of the dithionitereduced protein with L-serine results in a slight broadening of the feature at g ‫؍‬ 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.

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confidence: 74%

“…These are the enzymes from Peptostreptococcus asaccharolyticus (10), Clostridium propionicum (19), and Clostridium sticklandii (47). Each of these proteins is activated by iron and DTT; however, the nature of the iron-containing cofactor has been demonstrated only in the enzyme from P. asaccharolyticus.…”

Section: An Iron-sulfur Cluster In Serine Deaminasementioning

confidence: 99%

Escherichia coli L-Serine Deaminase Requires a [4Fe-4S] Cluster in Catalysis

Cicchillo¹,

Baker²,

Schnitzer³

et al. 2004

Journal of Biological Chemistry

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“…Since Fe appeared to be required to relieve NifL inhibition of NifA activity under anaerobic, nitrogen-limiting conditions in vivo, we performed a number of experiments in vivo and in vitro to determine whether NifL itself carried Fe or an Fe-S cluster. To test whether NifL (or any unidentified protein that participated in sensing oxygen or combined nitrogen in conjunction with NifL) carried an Fe-S cluster that was labile to superoxide (15,18,23), we overproduced Mn-SOD from E. coli in K. pneumoniae UN4495/pDT1-22 under aerobic nitrogen-limiting conditions to determine whether decreasing the concentration of superoxide would relieve the inhibitory effect of NifL on NifA activity.…”

Section: Resultsmentioning

confidence: 99%

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

Schmitz

Kustu

1996

J Bacteriol

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In Klebsiella pneumoniae, products of the nitrogen fixation nifLA operon regulate transcription of the other nif operons. NifA activates transcription by 54 -holoenzyme. In vivo, NifL antagonizes the action of NifA under aerobic conditions or in the presence of combined nitrogen. In contrast to a previous report, we show that depletion of iron (Fe) from the growth medium with the chelating agent o-phenanthroline (20 M) mimics aerobiosis or combined nitrogen in giving rise to inhibition of NifA activity even under anaerobic, nitrogenlimiting conditions. Adding back Fe in only twofold molar excess over phenanthroline restores NifA activity, whereas adding other metals fails to do so. By using strains that lack NifL, we showed that NifA activity itself does not require Fe and is not directly affected by phenanthroline. In the free-living diazotroph Klebsiella pneumoniae, expression of nitrogen fixation (nif) genes is regulated by the products of the nifLA operon (3,8,14,32). NifA activates transcription of all nif genes (except nifLA) by the alternative holoenzyme form of RNA polymerase 54 -holoenzyme. NifA binds to an upstream activation sequence (34) and contacts promoterbound 54 -holoenzyme by means of a DNA loop (9). It catalyzes the ATP-dependent isomerization of closed complexes between 54 -holoenzyme and a nif promoter to transcriptionally productive open complexes (25,35). NifL, which is a negative regulator, inhibits NifA activity in vivo in response to molecular oxygen and/or combined nitrogen (22,32).NifL from K. pneumoniae is composed of two domains separated by a hydrophilic interdomain linker (Q-linker [12]). We showed recently that the C-terminal domain is sufficient to inhibit transcriptional activation by NifA in vitro and in vivo (37). Thus, the inhibitory function of NifL appears to lie in its C-terminal domain, which presumably interacts with NifA. The N-terminal domain of NifL contains a region of homology (ϳ30% amino acid identity over 130 residues) to the product of the bat gene from Halobacterium halobium (19,47,50). Since Bat is an oxygen-responsive activator of the synthesis of bacterio-opsin, it has been proposed that the region of homology may be involved in oxygen sensing. The mechanism(s) by which NifL senses oxygen and/or combined nitrogen is not understood.The N-terminal domain of NifL contains one CysXXCys motif (Cys-184-Ala-Asp-Cys-187). The similarity of this motif to sequences involved in binding metal clusters in proteins such as ferrodoxins and rubredoxins (1, 4, 24) suggested a possible metal-binding role of this region in NifL (21). On the basis of this prediction, Henderson et al. (21) examined the influence of metal ion deficiency on NifL activity. They concluded that iron (Fe) deficiency reduced the inhibitory activity of NifL expressed at high levels in Escherichia coli.To further study the function of a possible metal ion or iron-sulfur (Fe-S) cluster in oxygen-sensing by NifL, we examined the effect of Fe deficiency on NifL inhibition of NifA activity in vivo in a stra...

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“…In general, the accumulation of waste products and active toxification are thought to be important determinants of environmental quality but were unlikely to factor into this experiment. Although ammonia, NH 3 , is a waste product of L-serine catabolism (Hofmeister and Buckler 1993), the concentrations produced, even if all L-serine was consumed, would be buffered as lower-toxicity ammonium ion NH 1 4 (only 1#10 3 mol/L would remain as ammonia given the pH and temperature conditions; Emerson et al 1975). Although Pseudomonas aeruginosa produces a wide variety of anticompetitor compounds, they are unlikely to be effective, as invader and resident share a common genetic background (Bucci et al 2011;Inglis et al 2011;Schoustra et al 2012).…”

Section: Discussionmentioning

confidence: 99%

Here’s to the Losers: Evolvable Residents Accelerate the Evolution of High-Fitness Invaders

Gifford¹,

Toll-Riera²,

Kojadinovic³

et al. 2015

The American Naturalist

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Recent work has shown that evolvability plays a key role in determining the long-term population dynamics of asexual clones. However, simple considerations suggest that the evolvability of a focal lineage of bacteria should also be influenced by the evolvability of its competitors. First, evolvable competitors should accelerate evolution by impeding the fixation of the focal lineage through a clonal interference-like mechanism. Second, evolvable competitors should increase the strength of selection by rapidly degrading the environment, increasing selection for adaptive mutations. Here we tested these ideas by allowing a high-fitness clone of the bacterium Pseudomonas aeruginosa to invade populations of two low-fitness resident clones that differ in their evolvability. Both competition from mutations in the resident lineage and environmental degradation lead to faster adaptation in the invader through fixing single mutations with a greater fitness advantage. The results suggest that competition from mutations in both the successful invader and the unsuccessful resident shapes the adaptive trajectory of the invader through both direct competition and indirect environmental effects. Therefore, to predict evolutionary outcomes, it will be necessary to consider the evolvability of all members of the community and the effects of adaptation on the quality of the environment. This is particularly relevant to mixed microbial communities where lineages differ in their adaptive potential, a common feature of chronic infections. abstract: Recent work has shown that evolvability plays a key role in determining the long-term population dynamics of asexual clones. However, simple considerations suggest that the evolvability of a focal lineage of bacteria should also be influenced by the evolvability of its competitors. First, evolvable competitors should accelerate evolution by impeding the fixation of the focal lineage through a clonal interference-like mechanism. Second, evolvable competitors should increase the strength of selection by rapidly degrading the environment, increasing selection for adaptive mutations. Here we tested these ideas by allowing a high-fitness clone of the bacterium Pseudomonas aeruginosa to invade populations of two low-fitness resident clones that differ in their evolvability. Both competition from mutations in the resident lineage and environmental degradation lead to faster adaptation in the invader through fixing single mutations with a greater fitness advantage. The results suggest that competition from mutations in both the successful invader and the unsuccessful resident shapes the adaptive trajectory of the invader through both direct competition and indirect environmental effects. Therefore, to predict evolutionary outcomes, it will be necessary to consider the evolvability of all members of the community and the effects of adaptation on the quality of the environment. This is particularly relevant to mixed microbial communities where lineages differ in their adaptive potential, a...

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L‐Serine and L‐threonine dehydratase from Clostridium propionicum Two enzymes with different prosthetic groups

Cited by 27 publications

References 33 publications

Escherichia coli L-Serine Deaminase Requires a [4Fe-4S] Cluster in Catalysis

Escherichia coli L-Serine Deaminase Requires a [4Fe-4S] Cluster in Catalysis

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

Here’s to the Losers: Evolvable Residents Accelerate the Evolution of High-Fitness Invaders

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