Thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the urease metallochaperone UreE

The two Ni2+ ions in the urease active site are delivered by the metallochaperone UreE, whose metal binding properties are central to the assembly of this metallocenter. Isothermal titration calorimetry (ITC) has been used to quantify the stoichiometry, affinity, and thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the well-studied C-terminal truncated H144*UreE from Klebsiella aerogenes, Ni2+ binding to the wild-type K. aerogenes UreE protein, and Ni2+ and Zn2+ binding to the wild-type UreE protein from Bacillus pasteurii. The stoichiometries and affinities obtained by ITC are in good agreement with previous equilibrium dialysis results, after differences in pH and buffer competition are considered, but the concentration of H144*UreE was found to have a significant effect on metal binding stoichiometry. While two metal ions bind to the H144*UreE dimer at concentrations <10 microM, three Ni2+ or Cu2+ ions bind to 25 microM dimeric protein with ITC data indicating sequential formation of Ni/Cu(H144*UreE)4 and then (Ni/Cu)2(H144*UreE)4, or Ni/Cu(H144*UreE)2, followed by the binding of four additional metal ions per tetramer, or two per dimer. The thermodynamics indicate that the latter two metal ions bind at sites corresponding to the two binding sites observed at lower protein concentrations. Ni2+ binding to UreE from K. aerogenes is an enthalpically favored process but an entropically driven process for the B. pasteurii protein, indicating chemically different Ni2+ coordination to the two proteins. A relatively small negative value of DeltaCp is associated with Ni2+ and Cu2+ binding to H144*UreE at low protein concentrations, consistent with binding to surface sites and small changes in the protein structure.     

Increased heterocyst frequency by patN disruption in Anabaena leads to enhanced photobiological hydrogen production at high light intensity and high cell density

The effects of increasing the heterocyst-to-vegetative cell ratio on the nitrogenase-based photobiological hydrogen production by the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were studied. Using the uptake hydrogenase-disrupted mutant (ΔHup) as the parent, a deletion-insertion mutant (PN1) was created in patN, known to be involved in heterocyst pattern formation and leading to multiple singular heterocysts (MSH) in Nostoc punctiforme strain ATCC 29133. The PN1 strain showed heterocyst differentiation but failed to grow in medium free of combined-nitrogen; however, a spontaneous mutant (PN22) was obtained on prolonged incubation of PN1 liquid cultures and was able to grow robustly on N₂. The disruption of patN was confirmed in both PN1 and PN22 by PCR and whole genome resequencing. Under combined-nitrogen limitation, the percentage of heterocysts to total cells in the PN22 filaments was 13–15 and 16–18% under air and 1% CO₂-enriched air, respectively, in contrast to the parent ΔHup which formed 6.5–11 and 9.7–13% heterocysts in these conditions. The PN22 strain exhibited a MSH phenotype, normal diazotrophic growth, and higher H₂ productivity at high cell concentrations, and was less susceptible to photoinhibition by strong light than the parent ΔHup strain, resulting in greater light energy utilization efficiency in H₂ production on a per unit area basis under high light conditions. The increase in MSH frequency shown here appears to be a viable strategy for enhancing H₂ productivity by outdoor cultures of cyanobacteria in high-light environments.

     

A structural perspective on the PP-loop ATP pyrophosphatase family

Derived from an ancient ATP-hydrolyzing Rossmann-like fold protein, members of the PP-loop ATP pyrophosphatase family feature an absolutely conserved P-loop-like “SxGxDS/T” motif used for binding and presenting ATP for substrate adenylylation (AMPylation). Since the first family member was reported more than 20 years ago, numerous representatives catalyzing very diverse reactions have been characterized both functionally and structurally. The availability of more than 100 high quality structures in the protein data bank provides an excellent opportunity to gain structural insights into the generally conserved catalytic mechanism and the uniqueness of the reactions catalyzed by family members. In this work, we conducted a thorough database search for the PP-loop ATP pyrophosphatase family members, resulting in the most comprehensive and up-to-date collection that includes 18 enzyme families. Structure comparison of representative family members allowed us to identify common structure features in the core catalytic domain, as well as four highly variable regions that define the unique chemistry for each enzyme family. The newly identified enzymes, particularly those from pathogens, warrant further research to enlarge the scope of this ever-expanding and highly diverse enzyme superfamily for use in potential bioengineering and biomedical applications.     

Mechanisms of metal ion incorporation into metalloproteins

Although the structure and function of protein metallocenters have been extensively characterized, much less is known about their assembly. Here, I describe several general strategies for metallocenter biosynthesis and provide literature precedents for each mechanism. The simplest mechanism involves reversible metal ion binding to amino acid ligands of the apo-protein. In a variation of this mechanism, the apo-protein first must be phosphorylated, carboxylated or otherwise covalently modified in order to create the metal ion binding site. Alternatively, passive metal ion binding may require the presence of an associated compound, such as a nucleotide, carbonate or inorganic sulfide, which is co-incorporated into the protein along with the metal ion. In addition, reversible binding may occur using a pre-formed organometallic cofactor such as a metal-tetrapyrrole. Electron transfer reactions are coupled to biosynthesis of certain metallocenters, i.e. oxidation or reduction of the metallocenter or apo-protein may be required prior to binding, or once bound the metallocenter may be oxidatively trapped in the protein. An effector molecule may bind to apo-protein to open up or stabilize the metallocenter binding site, then after the metallocenter is incorporated the effector molecule dissociates. A transferase or insertase protein first may bind the metallocenter and then incorporate it into the appropriate apo-protein. Finally, metal cofactors may be covalently attached to proteins. Regardless of the metallocenter biosynthetic mechanism, intracellular metal ion concentrations must be sufficient; hence, metal ion transport systems also are briefly discussed.