A possible mechanism for the <i>in vitro</i> activation of <scp>L</scp>-serine deaminase activity in <i>Escherichia coli</i> K12

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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“…LSD1 has been moderately characterized (13,15,16). It contains 454 amino acids, including 9 cysteines (13).…”

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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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“…L-SD is synthesized in an inactive form in E. coli (20). Several mutants have been isolated that are unable to make an active L-SD but make a protein that can be activated in vitro (20).The number of environmental factors and the variety of mechanisms affecting L-SD activity suggest that its metabolic role is important to the cell. Nonetheless, mutants deficient in L-SD show very little difference in phenotype from their parent strain (22, 28).…”

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confidence: 99%

“…L-SD is synthesized in an inactive form in E. coli (20). Several mutants have been isolated that are unable to make an active L-SD but make a protein that can be activated in vitro (20).…”

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confidence: 99%

A novel L-serine deaminase activity in Escherichia coli K-12

Newman

1991

J Bacteriol

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We demonstrate here that Escherichia coli K-12 synthesizes two different L-serine deaminases (L-SD) catalyzing the nonoxidative deamination of L-serine to pyruvate, one coded for by the previously described sdaA gene and a second, hitherto undescribed enzyme which we call L-SD2. A strain carrying a null mutation in sdaA made no detectable L-SD in minimal medium, but had activity in Luria broth. We describe a mutation, sdaX, which affects the regulation of L-SD2 and permits its expression in minimal medium, and an insertion mutation, sdaB, which abolishes L-SD2 activity completely. Both mutations lie near 60.5 min on the E. coli genetic map. The two L-SD enzymes have similar enzyme parameters, and both require posttranslational activation.Escherichia coli K-12, when grown in glucose-minimal medium, makes an enzyme, L-serine deaminase (L-SD), which is further induced in media with glycine and L-leucine added (15,24). This enzyme is even further induced by growth in Luria broth (LB) and is subject to complex regulation by at least two global regulatory systems (17,23). The enzyme is coded by the sdaA gene, which has been cloned and sequenced (28).L-SD activity is also affected by posttranslational control. L-SD is synthesized in an inactive form in E. coli (20). Several mutants have been isolated that are unable to make an active L-SD but make a protein that can be activated in vitro (20).The number of environmental factors and the variety of mechanisms affecting L-SD activity suggest that its metabolic role is important to the cell. Nonetheless, mutants deficient in L-SD show very little difference in phenotype from their parent strain (22, 28).We were surprised to find that, when grown in LB, a strain deficient in L-SD1 still had a considerable amount of L-SD activity, and we proposed that there might be a second L-SD, the synthesis of which was regulated differently from that of the first (22).In this work, we demonstrate the existence of this second enzyme in a strain carrying a null mutation in sdaA. L-SD2 is similar to L-SD1 in many of its parameters, but it clearly is different in structure and is encoded by a separate gene. MATERIALS AND METHODSCultures. The strains used in this study, all derivatives of E. coli K-12, are described in Table 1, as are the bacteriophages and plasmids.Growth conditions. The minimal medium used has been described previously (22). Because strains CU1008 and MEW1 and all their derivatives carry a deletion in ilvA, L-isoleucine and L-valine (50 p.g/ml each) were added to all media. When cells were grown for L-SD assays in LB, 0.5% glucose was added to completely repress the biodegradative L-threonine deaminase.SGL medium. Medium with a combination of L-serine, glycine, and L-leucine as the only carbon sources other than L-isoleucine and L-valine is called SGL medium, and strains * Corresponding author. unable to grow on this medium are termed SGL-(22). L-Serine, glycine, and L-leucine were usually provided at 2,000, 300, and 300 ,ug/ml, respectively.

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“…Nevertheless, there would be a sufficient number of cysteinyl residues to complex at least one iron-sulfur cluster. In addition, L-serine dehydratase of E. coli is similarly activated by FeZ+ and dithiothreitol (Newman et al, 1985). Hence, there is ample reason to believe that the enzyme from E. coli might also be a member of the class of iron-sulfur-dependent enzymes.…”

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confidence: 99%

“…These dehydratases are distinguished by their extreme instability and their ability to be protected, and in some cases activated, by substrate and/or competitive inhibitors (Alfoldi et al, 1968;Carter and Sagers, 1972;Morikawa et al, 1974;Gannon et al, 1977;Newman and Kapoor, 1980). Moreover, L-serine dehydratase from Clostridiuni acidi-urici (Carter and Sagers, 1972), E. coli (Newman et al, 1985). Lnctobacillus fermentum (Farias et al, 1991) and several other lactic acid bacteria (Farias et al, 1988) have been recognized to require activation by Fe".…”

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confidence: 99%

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

Hofmeister

Grabowski

Linder

1993

European Journal of Biochemistry

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L-Serine dehydratase from the Gram-positive bacterium Peptostreprococcus usuccharolyricus is novel in the group of enzymes deaminating 2-hydroxyamino acids in that it is an iron-sulfur protein and lacks pyridoxal phosphate [Grabowski, R. and Buckel, W. (1991) Eur. J. Biochem. 199, 89-941. It was proposed that this type of L-serine dehydratase is widespread among bacteria but has escaped intensive characterization due to its oxygen lability. Here, we present evidence that another Gram-positive bacterium, Clostridium pmpionicum, contains both an iron-sulfur-dependent L-serine dehydratase and a pyridoxal-phosphate-dependent L-threonine dehydratase. These findings support the notion that two independent mechanisms exist for the deamination of 2-hydroxyamino acids.L-Threonine dehydratase was purified 400-fold to apparent homogeneity and revealed as being a tetramer of identical subunits (m = 39 kDa). The purified enzyme exhibited a specific activity of 5 pkadmg protein and a K , for L-threonine of 7.7 mM. L-Serine ( K , = 380 mM) was also deaminated, the V/K, ratio, however, being 118-fold lower than the one for L-threonine. L-Threonine dehydratase was inactivated by borohydride, hydroxylamine and phenylhydrazine, all known inactivators of pyridoxal-phosphate-containing enzymes. Incubation with NaB3H, specifically labelled the enzyme. Activity of the phenylhydrazine-inactivated enzyme could be restored by pyridoxal phosphate.L-Serine dehydratase was also purified 400-fold, but its extreme instability did not permit purification to homogeneity. The enzyme was specific for L-serine ( K , = 5 mM) and was inhibited by L-cysteine (K, = 0.5 mM) and D-serine (K, = 8 mM). Activity was insensitive towards borohydride, hydroxylamine and phenylhydrazine but was rapidly lost upon exposure to air. Fez+ specifically reactivated the enzyme. L-Serine dehydratase was composed of two different subunits (a, m = 30kDa; p, m = 26kDa), their apparent molecular masses being similar to the ones of the two subunits of the iron-sulfur-dependent enzyme from Z? asuccharolyticus. Moreover, the N-terminal sequences of the small subunits from these two organisms were found to be 47% identical. In addition, 38% identity with the N-terminus of one of the two L-serine dehydratases of Escherichiu coli was detected.

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A possible mechanism for the in vitro activation of L-serine deaminase activity in Escherichia coli K12

Cited by 10 publications

References 15 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

A novel L-serine deaminase activity in Escherichia coli K-12

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

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