Iron—sulfur cluster‐containing l‐serine dehydratase from Peptostreptococcus asaccharolyticus: Correlation of the cluster type with enzymatic activity (original) (raw)
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European Journal of Biochemistry, 1993
l‐Serine dehydratase from the Gram‐positive bacterium Peptostreptococcus asaccharolyticus 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, 89–94]. 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 propionicum, 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= 39kDa). The purified enzyme exhibited a specific activity of 5 μkat/mg protein and a Km for l‐th...
Characterization of a L-serine dehydratase activity from Streptococcus faecalis
Le Lait, 1988
Streptococcus faecalis sp. produces pyruvate and ammonia from L-serine via a specifie L-serine dehydratase. The apparent Michaelis. constant for L-serine is 50 mM and the Vmax is 142 nmol of pyruvate formed per minute and per mg of protein. Maximum enzymatic activity is observed at 40 "C and pH 8 in 100 mM phosphate buffer. L-serine is the sole substrate. D-serine, L-threonine and glycine are competitive inhibitors, L-cysteine acts as a non-competitive inhibitor. The enzyme seems to be a very labile protein ; it is inactivated by dilution, dialysis and temperature. Pyridoxal phosphate is not required for maximum activity. L-serine dehydratase is strongly inhibited by Cu 2 +, Zn 2 +, Hg 2 +, p-chloromercuribenzoate, Tris, borate and acetate. The enzymatic activity is stimulated by Fe 2 + ions.
Journal of Biological Chemistry, 1998
The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase). A 5-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform.
Biochemistry, 2003
The last step of the biosynthesis of fosfomycin, a clinically useful antibiotic, is the conversion of (S)-2-hydroxypropylphosphonic acid (HPP) to fosfomycin. Since the ring oxygen in fosfomycin has been shown in earlier feeding experiments to be derived from the hydroxyl group of HPP, this oxirane formation reaction is effectively a dehydrogenation process. To study this unique CO bond formation step, we have overexpressed and purified the desired HPP epoxidase. Results reported herein provided initial biochemical evidence revealing that HPP epoxidase is an iron-dependent enzyme and that both NAD(P)H and a flavin or flavoprotein reductase are required for its activity. The 2 K EPR spectrum of oxidized iron-reconstituted fosfomycin epoxidase reveals resonances typical of S) 5 / 2 Fe(III) centers in at least two environments. Addition of HPP causes a redistribution with the appearance of at least two additional species, showing that the iron environment is perturbed. Exposure of this sample to NO elicits no changes, showing that the iron is nearly all in the Fe(III) state. However, addition of NO to the Fe(II) reconstituted enzyme that has not been exposed to O 2 yields an intense EPR spectrum typical of an S) 3 / 2 Fe(II)-NO complex. This complex is also heterogeneous, but addition of substrate converts it to a single, homogeneous S) 3 / 2 species with a new EPR spectrum, suggesting that substrate binds to or near the iron, thereby organizing the center. The fact that NO binds to the ferrous center suggests O 2 can also bind at this site as part of the catalytic cycle. Using purified epoxidase and 18 O isotopic labeled HPP, the retention of the hydroxyl oxygen of HPP in fosfomycin was demonstrated. While ether ring formation as a result of dehydrogenation of a secondary alcohol has precedence in the literature, these catalyses require R-ketoglutarate for activity. In contrast, HPP epoxidase is R-ketoglutarate independent. Thus, the cyclization of HPP to fosfomycin clearly represents an intriguing conversion beyond the scope entailed by common biological epoxidation and CO bond formation.
Journal of Biological Inorganic Chemistry, 2004
We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126±2 kDa composed of two subunits, a=93±3 kDa and b=32±2 kDa, which contains 6±1 Fe/molecule, 0.4±0.1 Mo/molecule, 0.3±0.1 W/molecule, and 1.3±0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5-40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d 1 configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme. Keywords Electron paramagnetic resonance AE Formate dehydrogenase AE Magnetic interactions AE Molybdenum-containing enzymes AE Tungsten-containing enzymes Abbreviations AOR aldehyde oxidoreductase AE FDH formate dehydrogenase AE NAP periplasmic nitrate reductase AE SRB sulfate-reducing bacteria
Iron-sulfur proteins: Recent developments in the field
Experientia, 1982
Iron-sulfur clusters in proteins are now recognized as among the main types of electron-transferring groups in biological systems, besides heme and flavins. Recent developments have brought forth a better understanding about the ways the protein environment modulates the potential of the cluster by placing the cluster in a more or less hydrophobic surrounding. Refinement in models, extensive studies on the kinetics of electron transfer (e.g. by measurement of the electronic spin lattice relaxation time) and the introduction of novel spectroscopic methods (EXAFS, magnetic CD and others) in the elucidation of structures in various systems are among the main developments. Other advances include EPR studies of the spatial orientation of Fe-S centers in complex membraneous systems (e.g. in mitochondria) and the recent elucidation of the nature of center X in photosystem I by M6ssbauer-spectroscopy. M6ssbauer studies have also been described on a number of Fe-S proteins (nitrogenase, aconitase, some ferredoxins, etc.) and revealed the existence of novel structures that enlarged the number of known basic units of Fe-S centers. These advances include: 1. the discovery of a novel non-heme Fe-protein (called desulforedoxin) of the rebredoxin type, 2. the elucidation of the nitrogenase Fe-S centers and the nitrogenase cofactor and 3. the discovery of a three-iron cluster in several enzyme s and some ferredoxins. The latter 3-Fe cluster seems capable of being converted into a classical 4-Fe cluster under appropriate conditions, a phenomenon that plays a role in activation-deactivation of some enzymes (e.g. aconitase). It is now recognized that some iron-sulfur clusters may be involved in systems devoided of any oxydation-reduction reaction and may act as sensors of the surrounding redox potential, triggering the activation/deactivation of an enzyme (cf. e.g. aconitase).
Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931
Current Microbiology, 1991
L-Serine dehydratase from Lactobacillus.fermentum was purified 100-fold. It was stabilized by the presence of 1 mM L-cysteine in 50 mM phosphate buffer. M r = 150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (Mr = 40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent Km for L-serine was 65 mM. Fe + + was required for the enzymatic activity, and the apparent K m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal x mol 1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5'-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 p.moles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.
Cluster characterization in iron-sulfur proteins by magnetic circular dichroism
Proceedings of the National Academy of Sciences, 1978
We report magnetic circular dichroism (MCD) spectra of 4-Fe iron-sulfur clusters in the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP), Bacillus stearothernophilus ferredoxin and Clostridium pasteurianum ferredoxin. The MCD is found to vary significantly with cluster oxidation state but is relatively insensitive to the nature of the protein. The spectra obtained are compared with the corresponding spectra of iron-sulfur proteins containing 2-Fe clusters. It is concluded that MCD is useful for the characterization of iron-sulfur cluster type and oxidation state in iron-sulfur proteins and is superior for this purpose to absorption and nat-