Metal cofactors of lysine-2,3-aminomutase.

Lysine-2,3-aminomutase from Clostridium SB4 contains iron and sulfide in equimolar amounts, as well as cobalt, zinc, and copper. The iron and sulfide apparently constitute an Fe-S cluster that is required as a cofactor of the enzyme. Although no B12 derivative can be detected, enzyme-bound cobalt is a cofactor; however, the zinc and copper bound to the enzyme do not appear to play a role in its catalytic activity. These conclusions are supported by the following facts reported in this paper. Purification of the enzyme under anaerobic conditions increases the iron and sulfide content. Lysine-2,3-aminomutase purified from cells grown in media supplemented with added CoCl2 contains higher levels of cobalt and correspondingly lower levels of zinc and copper relative to enzyme from cells grown in media not supplemented with cobalt. The specific activity of the purified enzyme increases with increasing iron and sulfide content, and it also increases with increasing cobalt and with decreasing zinc and copper content. The zinc and copper appear to occupy cobalt sites under conditions of insufficient cobalt in the growth medium, and they do not support the activity of the enzyme. The best preparations of lysine-2,3-aminomutase obtained to date exhibit a specific activity of approximately 23 units/mg of protein and contain about 12 g atoms of iron and of sulfide per mol of hexameric enzyme. These preparations also contain 3.5 g atoms of cobalt per mol, but even the best preparations contain small amounts of zinc and copper. The sum of cobalt, zinc, and copper in all preparations analyzed to date corresponds to 5.22 +/- 0.75 g atoms per mol of enzyme. An EPR spectrum of the enzyme as isolated reveals a signal corresponding to high spin Co(II) at temperatures below 20 K. The signal appears as a partially resolved 59Co octet centered at an apparent g value of 7. The 59Co hyperfine splitting (approximately 35 G) is prominent at 4.2 K. These findings show that lysine-2,3-aminomutase requires Fe-S clusters and cobalt as cofactors, in addition to the known requirement for pyridoxal 5'-phosphate and S-adenosylmethionine.     

The high potential iron-sulfur cluster of aconitase is a binuclear iron-sulfur cluster.

It has been reported (Ruzicka, F.J., and Beinert, H. (1978) J. Biol. Chem. 253, 2514-2517) that aconitase in the oxidized state, as isolated, shows an electron paramagnetic resonance signal centered at g = 2.01, typical of high potential iron-sulfur proteins. Since the magnetic state corresponding to this signal has thus far only been found in tetranuclear iron-sulfur clusters in model compounds and proteins, it could be expected that aconitase also contains a [4Fe-4S] cluster. We show here that core extrusion, in the presence of hexamethylphosphoramide and o-xylyl-alpha,alpha'-dithiol and subsequent ligand exchange with p-trifluoromethylbenzenethiol yield absorption spectra typical of binuclear iron-sulfur clusters. According to the absorbance measured, the concentration of the extruded [2Fe-2S] cluster quantitatively accounts for the iron-sulfur content of the preparations examined. Preliminary studies of the 19F nuclear magnetic resonance spectrum obtained on extrusion with p-trifluoromethylbenzenethiol confirm the presence of a binuclear cluster in aconitase.     

The Radical SAM Superfamily

ABSTRACT

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe–4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe–4S]^{1 +} –SAM complex to [4Fe–4S]^{2 +}–Met and the 5′ -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.     

Phe(475) and Glu(446) but not Ser(445) participate in ATP-binding to the alpha-subunit of Na(+)/K(+)-ATPase

The ATP-binding site of Na(+)/K(+)-ATPase is localized on the large cytoplasmic loop of the alpha-subunit between transmembrane helices H(4) and H(5). Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser(445), Glu(446), and Phe(475) were proposed to be close to the binding pocket. Replacement of Phe(475) with Trp and Glu(446) with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser(445) with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser(445) is close to the binding site, although it does not participate in binding.     

Northern Bobwhite (Colinus virginianus) Mitochondrial Population Genomics Reveals Structure,Divergence, and Evidence for Heteroplasmy

Herein, we evaluated the concordance of population inferences and conclusions resulting from the analysis of short mitochondrial fragments (i.e., partial or complete D-Loop nucleotide sequences) versus complete mitogenome sequences for 53 bobwhites representing six ecoregions across TX and OK (USA). Median joining (MJ) haplotype networks demonstrated that analyses performed using small mitochondrial fragments were insufficient for estimating the true (i.e., complete) mitogenome haplotype structure, corresponding levels of divergence, and maternal population history of our samples. Notably, discordant demographic inferences were observed when mismatch distributions of partial (i.e., partial D-Loop) versus complete mitogenome sequences were compared, with the reduction in mitochondrial genomic information content observed to encourage spurious inferences in our samples. A probabilistic approach to variant prediction for the complete bobwhite mitogenomes revealed 344 segregating sites corresponding to 347 total mutations, including 49 putative nonsynonymous single nucleotide variants (SNVs) distributed across 12 protein coding genes. Evidence of gross heteroplasmy was observed for 13 bobwhites, with 10 of the 13 heteroplasmies involving one moderate to high frequency SNV. Haplotype network and phylogenetic analyses for the complete bobwhite mitogenome sequences revealed two divergent maternal lineages (d _XY = 0.00731; F _ST = 0.849; P < 0.05), thereby supporting the potential for two putative subspecies. However, the diverged lineage (n = 103 variants) almost exclusively involved bobwhites geographically classified as Colinus virginianus texanus, which is discordant with the expectations of previous geographic subspecies designations. Tests of adaptive evolution for functional divergence (MKT), frequency distribution tests (D, F _S) and phylogenetic analyses (RAxML) provide no evidence for positive selection or hybridization with the sympatric scaled quail (Callipepla squamata) as being explanatory factors for the two bobwhite maternal lineages observed. Instead, our analyses support the supposition that two diverged maternal lineages have survived from pre-expansion to post-expansion population(s), with the segregation of some slightly deleterious nonsynonymous mutations.     

Capillary isoelectric focusing--useful tool for detection of the biofilm formation in Staphylococcus epidermidis

The biofilm formation is an important factor of S. epidermidis virulence. Biofilm-positive strains might be clinically more important than biofilm-negative ones. Unlike biofilm-negative staphylococci, biofilm-positive staphylococci are surrounded with an extracellular polysaccharide substance. The presence of this substance on the surface can affect physico-chemical properties of the bacterial cell, including surface charge. 73 S. epidermidis strains were examined for the presence of ica operon, for the ability to form biofilm by Christensen test tube method and for the production of slime by Congo red agar method. Isoelectric points (pI) of these strains were determined by means of Capillary Isoelectric Focusing. The biofilm negative strains focused near pI value 2.3, while the pI values of the biofilm positive strains were near 2.6. Isoelectric point is a useful criterion for the differentiation between biofilm-positive and biofilm-negative S. epidermidis strains.