Intracellular nickel is required by Escherichia coli as a cofactor for a number of enzymes and is necessary for anaerobic respiration. However, high concentrations of nickel are toxic, so both import and export systems have evolved to control the cellular level of the metal. The nik operon in E. coli encodes a nickel-uptake system that includes the periplasmic nickel-binding protein NikA. The crystal structures of wild-type NikA both bound to nickel and in the apo form have been solved previously. The liganded structure appeared to show an unusual interaction between the nickel and the protein in which no direct bonds are formed. The highly unusual nickel coordination suggested by the crystal structure contrasted strongly with earlier X-ray spectroscopic studies. The known nickel-binding site has been probed by extensive mutagenesis and isothermal titration calorimetry and it has been found that even large numbers of disruptive mutations appear to have little effect on the nickel affinity. The crystal structure of a binding-site mutant with nickel bound has been solved and it is found that nickel is bound to two histidine residues at a position distant from the previously characterized binding site. This novel site immediately resolves the conflict between the crystal structures and other biophysical analyses. The physiological relevance of the two binding sites is discussed.
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"This result also applied to the structure determined by Heddle and collaborators. Indeed, in 2007, these authors revised their NikA structure and agreed with our previous interpretation . Therefore, all these studies suggested the existence of a EDTA-like natural chelator involved in nickel binding. "
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli require nickel for the synthesis of [NiFe] hydrogenases under anaerobic growth conditions. Nickel import depends on the specific ABC-transporter NikABCDE encoded by the nik operon, which deletion causes the complete abolition of hydrogenase activity. We have previously postulated that the periplasmic binding protein NikA binds a natural metallophore containing three carboxylate functions that coordinate a Ni(II) ion, the fourth ligand being His416, the only direct metal-protein contact, completing a square-planar coordination for the metal. The crystal structure of the H416I mutant showed no electron density corresponding to a metal-chelator complex. In vivo experiments indicate that the mutation causes a significant decrease in nickel uptake and hydrogenase activity. These results confirm the essential role of His416 in nickel transport by NikA.
"Cherrier et al. suggested that NikA binds Ni chelated by a small organic molecule, such as butane-1,2,4-tricarboxylate (BTC), and that some residues, including Tyr402, Arg137, Arg97 and His416, form a binding site that is involved in the BTC-Ni-NikA interaction . On the other hand, Addy and coworkers showed that Ni may bind E. coli NikA without chelators and is bound to two histidine residues (His56 and His442, although not conserved in other NikA proteins) at a position distant from the previously characterized binding site . Here, the presence of the majority of these residues was used to help predict NikA proteins. "
[Show abstract][Hide abstract] ABSTRACT: Nickel (Ni) and cobalt (Co) are trace elements required for a variety of biological processes. Ni is directly coordinated by proteins, whereas Co is mainly used as a component of vitamin B12. Although a number of Ni and Co-dependent enzymes have been characterized, systematic evolutionary analyses of utilization of these metals are limited.
We carried out comparative genomic analyses to examine occurrence and evolutionary dynamics of the use of Ni and Co at the level of (i) transport systems, and (ii) metalloproteomes. Our data show that both metals are widely used in bacteria and archaea. Cbi/NikMNQO is the most common prokaryotic Ni/Co transporter, while Ni-dependent urease and Ni-Fe hydrogenase, and B12-dependent methionine synthase (MetH), ribonucleotide reductase and methylmalonyl-CoA mutase are the most widespread metalloproteins for Ni and Co, respectively. Occurrence of other metalloenzymes showed a mosaic distribution and a new B12-dependent protein family was predicted. Deltaproteobacteria and Methanosarcina generally have larger Ni- and Co-dependent proteomes. On the other hand, utilization of these two metals is limited in eukaryotes, and very few of these organisms utilize both of them. The Ni-utilizing eukaryotes are mostly fungi (except saccharomycotina) and plants, whereas most B12-utilizing organisms are animals. The NiCoT transporter family is the most widespread eukaryotic Ni transporter, and eukaryotic urease and MetH are the most common Ni- and B12-dependent enzymes, respectively. Finally, investigation of environmental and other conditions and identity of organisms that show dependence on Ni or Co revealed that host-associated organisms (particularly obligate intracellular parasites and endosymbionts) have a tendency for loss of Ni/Co utilization.
Our data provide information on the evolutionary dynamics of Ni and Co utilization and highlight widespread use of these metals in the three domains of life, yet only a limited number of user proteins.
[Show abstract][Hide abstract] ABSTRACT: In this paper, a fully integrated 2 GHz low phase noise LC-tank VCO, designed in 0.35 μm CMOS technology is presented and analyzed. The phase noise of the VCO has been greatly reduced by means of integrated source degeneration inductors. The phase noise achieved is -108, -125 dBc/Hz and -139 dBc/Hz at 100 kHz, 600 kHz and 3 MHz offsets respectively from the carrier frequency of 2 GHz, with 2.5 V power supply voltage and giving a power consumption of 10.35 mW. A 20.3% wide tuning range is achieved by means of PMOS varactors and a degeneration resistor. The tunability of the designed oscillator covers 380 MHz, from 1.68 GHz up to 2.06 GHz with a VCO gain of 280 MHz/V. The proposed VCO is fully operational under variable conditions including temperature and supply voltage variations from -40°C to 85°C and 2.5 V ±10%, respectively.
Microelectronics, 2004. ICM 2004 Proceedings. The 16th International Conference on; 01/2005