Catalytic Metal Binding Research Articles

Identification of virulence-associated factors in Vibrio parahaemolyticus with special reference to moonlighting protein: a secretomics study.

Vibrio parahaemolyticus causes seafood-borne gastroenteritis infection in human which can even lead to death. The pathogenic strain of V. parahaemolyticus secretes different types of virulence factors thatare directly injected into the host cell by a different type of secretion system which helps bacteria to establish its own ecological niche within the organism. Therefore, the aim of this study was to isolate the extracellular secreted proteins from the trh positive strain of V. parahaemolyticus and identify them using two-dimensional gel electrophoresis and MALDI-TOFMS/MS. Seventeen different cellular proteins viz, Carbamoyl-phosphate synthase, 5-methyltetrahydropteroyltriglutamate, tRNA-dihydrouridine synthase, Glycerol-3-phosphate dehydrogenase, Orotidine 5'-phosphate decarboxylase, Molybdenum import ATP-binding protein, DnaJ, DNA polymerase IV, Ribosomal RNA small subunit methyltransferase G, ATP synthase subunit delta and gamma, Ribosome-recycling factor, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, tRNA pseudouridine synthase B, Ditrans, polycis-undecaprenyl-diphosphate synthase, Oxygen-dependent coproporphyrinogen-III oxidase, and Peptide deformylase 2 were identified whichare mainly involved in different metabolic and biosynthetic pathways. Furthermore, the molecular function of the identified proteins were associated with catalytic activity, ligase activity, transporter, metal binding, and ATP synthase when they are intercellular. However, to understand the importance of these secreted proteins in the infection and survival of bacteria inside the host cell, pathogen-host protein-protein interactions (PPIs) were carried out which identified the association of eight secreted proteins with 41 human proteins involved in different cellular pathways, including ubiquitination degradation, adhesion, inflammation, immunity, and programmed cell death. The present study provides unreported strategies on host-cell environment's survival and adaptation mechanisms for the successful establishment of infections and intracellular propagation.

Identification of an Additional Metal-Binding Site in Human Dipeptidyl Peptidase III.

Dipeptidyl peptidase III (DPP III, EC 3.4.14.4) is a monozinc metalloexopeptidase that hydrolyzes dipeptides from the N-terminus of peptides consisting of three or more amino acids. Recently, DPP III has attracted great interest from scientists, and numerous studies have been conducted showing that it is involved in the regulation of various physiological processes. Since it is the only metalloenzyme among the dipeptidyl peptidases, we considered it important to study the process of binding and exchange of physiologically relevant metal dications in DPP III. Using fluorimetry, we measured the Kd values for the binding of Zn2+, Cu2+, and Co2+ to the catalytic site, and using isothermal titration calorimetry (ITC), we measured the Kd values for the binding of these metals to an additional binding site. The structure of the catalytic metal's binding site is known from previous studies, and in this work, the affinities for this site were calculated for Zn2+, Cu2+, Co2+, and Mn2+ using the QM approach. The structures of the additional binding sites for the Zn2+ and Cu2+ were also identified, and MD simulations showed that two Cu2+ ions bound to the catalytic and inhibitory sites exchanged less frequently than the Zn2+ ions bound to these sites.

RNA Dependent RNA Polymerases of Severe Acute Respiratory Syndrome-Related Coronaviruses- An Insight into their Active Sites and Mechanism of Action

Aim: To analyze the RNA-dependent RNA polymerases (RdRps) of Severe Acute Respiratory Syndrome (SARS)-related coronaviruses (CoVs) to find out the conserved motifs, metal binding sites and catalytic amino acids and propose a plausible mechanism of action for these enzymes, using SARS-CoV-2 RdRp as a model enzyme.
 Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis (SDM), X-ray crystallographic and cryo-Electron microscopic (cryo-EM) data were analyzed.
 Methodology: Bioinformatics, Biochemical, Site-directed mutagenesis, X-ray crystallographic and cryo-EM data of these enzymes from RNA viral pathogens were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the RdRps.
 Results: Multiple sequence alignment (MSA) of RdRps from different SARS-related CoVs show a large number of highly conserved motifs among them. Though the RdRp from the Middle Eastern Respiratory syndrome (MERS)-CoV differed in many conserved regions yet the active site regions are completely conserved. Possible catalytic regions consist of an absolutely conserved amino acid K, as in single subunit (SSU) RNA polymerases and most of the DNA dependent DNA polymerases (DdDps). The invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU DNA dependent RNA polymerases (DdRps), prokaryotic multi-subunit (MSU) DdRps and DdDps is also highly conserved in the RdRps of SARS-CoVs. The universal metal binding motif –GDD- and an additional motif–SDD- are also found in all SARS-CoV RdRps. In stark contrast, the (–) strand RNA viral pathogens like Ebola, rabies, etc. use –GDN- rather than –GDD- for catalytic metal binding. An invariant YA pair (instead of an YG pair) is found in the primases of the SARS-CoVs. The SARS-CoVs RdRps and primases exhibit very similar active site and catalytic regions with almost same distance conservations between the template binding YG/YA pair and the catalytic K. In SARS-CoV RdRps an invariant R is placed at -5 which is shown to play a role in nucleoside triphosphate (NTP) selection and is in close agreement with SSU DdRps (viral family) and DdDps. In primases no such invariant R/K/H is found very close to the catalytic K in the downstream region, as found in RdRp and Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domains. An invariant YA pair is placed in the NiRAN domain instead of an YG pair, and an invariant H is placed at -5 position. Moreover, the Zn binding motif with the completely conserved Cs and a few DxD/DxxD type metal binding motifs are found in the RdRps and NiRAN domain. However, the primases contained only the DXD type metal binding motifs.
 Conclusions: The SARS and SARS-related CoV RdRps are very similar as large peptide regions are highly conserved among them. The closer identity between the RdRps of palm civet-CoV and SARS-CoV (CoV-1) suggest their possible link as found between their spike proteins also. The invariant YG and KL pairs may play a role in template binding and catalysis in SARS-CoV RdRps as reported in DdDps. An additional invariant –YAN- motif found in SARS-CoV RdRps may play a crucial role in nucleotide discrimination.

Binding and enzymatic properties of Ageritin, a fungal ribotoxin with novel zinc-dependent function

Ribotoxins are fungal proteins that serve as weapons against parasites and insects. They are strongly toxic due to their ability to enter host cells and inactivate ribosomes. Ageritin is the prototype of a new ribotoxin-like protein family present in basidiomycetes. We demonstrate that this enzyme has peculiar binding and enzymatic features. Different from other ribotoxins, its ribonucleolytic activity requires the presence of divalent cations, with a maximum activation in the presence of zinc ions, for which Ageritin exhibits the strongest affinity of binding. We modeled the catalytic metal binding site of Ageritin, made of the putative triad Asp68, Asp70 and His77. This report highlights that Ageritin has the structure and function of an RNase but a Mg2+/Zn2+-dependent mechanism of action, a new finding for ribotoxins. As a zinc-dependent toxin, Ageritin can be classified among the arsenal of zinc-binding proteins involved in fungal virulence.

The Loss and Gain of Functional Amino Acid Residues Is a Common Mechanism Causing Human Inherited Disease.

Elucidating the precise molecular events altered by disease-causing genetic variants represents a major challenge in translational bioinformatics. To this end, many studies have investigated the structural and functional impact of amino acid substitutions. Most of these studies were however limited in scope to either individual molecular functions or were concerned with functional effects (e.g. deleterious vs. neutral) without specifically considering possible molecular alterations. The recent growth of structural, molecular and genetic data presents an opportunity for more comprehensive studies to consider the structural environment of a residue of interest, to hypothesize specific molecular effects of sequence variants and to statistically associate these effects with genetic disease. In this study, we analyzed data sets of disease-causing and putatively neutral human variants mapped to protein 3D structures as part of a systematic study of the loss and gain of various types of functional attribute potentially underlying pathogenic molecular alterations. We first propose a formal model to assess probabilistically function-impacting variants. We then develop an array of structure-based functional residue predictors, evaluate their performance, and use them to quantify the impact of disease-causing amino acid substitutions on catalytic activity, metal binding, macromolecular binding, ligand binding, allosteric regulation and post-translational modifications. We show that our methodology generates actionable biological hypotheses for up to 41% of disease-causing genetic variants mapped to protein structures suggesting that it can be reliably used to guide experimental validation. Our results suggest that a significant fraction of disease-causing human variants mapping to protein structures are function-altering both in the presence and absence of stability disruption.

Influence of chemical denaturants on the activity, fold and zinc status of anthrax lethal factor

Anthrax lethal factor (LF) is a zinc-dependent endopeptidase which, through a process facilitated by protective antigen, translocates to the host cell cytosol in a partially unfolded state. In the current report, the influence of urea and guanidine hydrochloride (GdnHCl) on LF׳s catalytic function, fold and metal binding was assessed at neutral pH. Both urea and GdnHCl were found to inhibit LF prior to the onset of unfolding, with the inhibition by the latter denaturant being a consequence of its ionic strength. With the exception of demetallated LF (apoLF) in urea, unfolding, as monitored by tryptophan fluorescence spectroscopy, was found to follow a two-state (native to unfolded) mechanism. Analysis of the metal status of LF with 4-(2-pyridylazoresorcinol) (PAR) following urea or GdnHCl exposure suggests the enzyme to be capable of maintaining its metal ion passed the observed unfolding transition in a chelator-inaccessible form. Although an increase in the concentration of the denaturants eventually allowed the chelator access to the protein׳s zinc ion, such process is not correlated with the release of the metal ion. Indeed, significant dissociation of the zinc ion from LF was not observed even at 6M urea, and only high concentrations of GdnHCl (>3M) were capable of inducing the release of the metal ion from the protein. Hence, the current study demonstrates not only the propensity of LF to tightly bind its zinc ion beyond the spectroscopically determined unfolding transition, but also the utility of PAR as a structural probe.

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu(2+) (K(d) = 55 nM; from isothermal titration calorimetry) and higher preference for Cu(1+) (K(d) ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu(2+) of 275 mV using a thermodynamic cycle). Strong binding of Cu(1+) was also reflected in a reduction in the pK(a) values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a (2)H/(1)H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates.

Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5′ processing

Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana. PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world.

Regulatory Control or Oxidative Damage? Proteomic Approaches to Interrogate the Role of Cysteine Oxidation Status in Biological Processes

Oxidation is a double-edged sword for cellular processes and its role in normal physiology, cancer and aging remains only partially understood. Although oxidative stress may disrupt biological function, oxidation-reduction (redox) reactions in a cell are often tightly regulated and play essential physiological roles. Cysteines lie at the interface between these extremes since the chemical properties that make specific thiols exquisitely redox-sensitive also predispose them to oxidative damage by reactive oxygen or nitrogen species during stress. Thus, these modifications can be either under reversible redox regulatory control or, alternatively, a result of reversible or irreversible oxidative damage. In either case, it has become increasingly important to assess the redox status of protein thiols since these modifications often impact such processes as catalytic activity, conformational alterations, or metal binding. To better understand the redox changes that accompany protein cysteine residues in complex biological systems, new experimental approaches have been developed to identify and characterize specific thiol modifications and/or changes in their overall redox status. In this review, we describe the recent technologies in redox proteomics that have pushed the boundaries for detecting and quantifying redox cysteine modifications in a cellular context. While there is no one-size-fits-all analytical solution, we highlight the rationale, strengths, and limitations of each technology in order to effectively apply them to specific biological questions. Several technological limitations still remain unsolved, however these approaches and future developments play an important role toward understanding the interplay between oxidative stress and redox signaling in health and disease.

X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides

We have determined the X-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O-helix, but only the correct base pairing between the +2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groups of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction.

Evidence of Adaptability in Metal Coordination Geometry and Active-Site Loop Conformation among B1 Metallo-β-lactamases,

Subclass B1 beta-lactamases are Zn(II)-dependent hydrolases that confer bacterial resistance to most clinically useful beta-lactam antibiotics. The enzyme BcII from Bacillus cereus is a prototypical enzyme that belongs to this group, the first Zn(II)-dependent beta-lactamase to be discovered. Crucial aspects of the BcII catalytic mechanism and metal binding mode have been assessed mostly on the Co(II)-substituted surrogate. Here we report a high-resolution structure of Co(II)-BcII, revealing a metal coordination geometry identical to that of the native zinc enzyme. In addition, a high-resolution structure of the apoenzyme, together with structures with different degrees of metal occupancy and oxidation levels of a conserved Cys ligand, discloses a considerable mobility of two loops containing four metal ligands (namely, regions His116-Arg121 and Gly219-Cys221). This flexibility is expected to assist in the structural rearrangement of the metal sites during catalytic turnover, which, along with the coordination geometry adaptability of Zn(II) ions, grants the interaction with a variety of substrates, a characteristic feature of B1 metallo-beta-lactamases.

Characterisation of the first wheat ( Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase resembling mammalian counterparts

A formerly unknown nucleotide pyrophosphatase/phosphodiesterase (NPP) of wheat ( Triticum aestivum L.) (TaNPP) was recombinantly produced in Pichia pastoris (TaNPPr) following assembly of the sequence out of wheat ESTs using sequences of known NPPs. Simultaneously, a phosphodiesterase was purified to homogeneity from wheat germs, characterised and identified as TaNPP. TaNPP contains the highly conserved catalytic substrate and metal binding residues and displays properties [high pH optimum, glycosylation, high thermostability and inhibition by EDTA and adenosine 5′-monophosphate (AMP)] similar to those of mammalian enzymes characterised earlier. The sequence of TaNPP includes that of a putative transmembrane domain, a characteristic of most NPPs. Both recombinant and native TaNPP partially occur as oligomeric proteins on SDS PAGE. Upon addition of DTT, both migrate as monomeric proteins. Part of the native wheat protein even occurs in its truncated form, thus lacking the transmembrane region. Out of a range of tested natural substrates, TaNPP had the highest affinity towards adenine containing nucleotides. While Michaelis-Menten kinetics were valid in the low substrate concentration range, at higher concentrations, they were no longer applicable. TaNPP shows no similarity to the recently characterised rice and barley enzymes and, is hence, one of the first characterised plant NPPs resembling mammalian NPPs.

In the land of the rising sun with the COP9 signalosome and related Zomes

Zomes V: The Fifth International Symposium on the COP9 signalosome, Proteasome and eIF3 took place between 11 and 14 November 2008, in Yokohama, Japan, and was organized by J‐Y. Kato, M. Matsui, K. Iwai, K. Tanaka, M.Glickman, N. Wei & D. Chamovitz. ![][1] See Glossary for abbreviations used in this article Three macromolecular complexes—eIF3, the 26S proteasome lid and the CSN, referred to herein as ‘Zomes’—share a unique molecular architecture and have an impact on proteins from synthesis to degradation. In higher eukaryotes, each complex consists of six subunits carrying a PCI domain and two subunits carrying an MPN domain, and each complex performs a distinct cellular function: eIF3 is involved in protein translation; the proteasome lid participates in protein degradation; and the CSN seems to have multiple functions related to the other two complexes, most of which regulate protein stability. However, despite years of research, the functional relationships between the three Zomes, their molecular architecture, the contribution of the individual subunits to the complexes, and the precise function and regulation of each have remained poorly understood. Aspects of these questions were discussed at Zomes V: The Fifth International Symposium on the COP9 signalosome, Proteasome and eIF3, which brought together more than 100 investigators from diverse fields working with various biological systems. Here, we highlight some of the main issues that were discussed at the conference. Among the three Zomes, the proteasome lid and the CSN share the highest homology (Fig 1): for each subunit of the proteasome lid, there is a paralogous subunit in the CSN complex. For example, Csn5 and Rpn11 are particularly close paralogues of one another, with both having a catalytic metal binding metalloprotease motif, MPN+/JAMM, which is responsible for the isopeptidase activity of each intact complex. Functionally, Rpn11 provides ubiquitin deconjugating activity to the proteasome, whereas … [1]: /embed/graphic-1.gif

Harnessing natures ability to control metal ion coordination geometry using de novo designed peptides

Advances in protein chemistry and molecular and structural biology have empowered modern chemists to build complex biological architectures using a "first principles" approach, which is known as de novo protein design. In this Perspective we demonstrate how simple three-stranded alpha-helical constructs can be prepared by the sole consideration of the primary amino acid sequence of a peptide. With these well defined systems, we then demonstrate that metal binding cavities can be carved out of the hydrophobic cores of these aggregates in order to bind metal ions such as cadmium with well defined coordination geometries. Examples will be given of homoleptic CdS(3) complexes, CdS(3)O sites and proteins which contain equilibrium mixtures of these two species. We will provide a description of a strategy that allows us to build heterochromic peptides (small proteins that complex two metals in nearly identical environments but which result in different physical properties and allow for metal site selectivity). We conclude with a new class of designed peptides, diastereopeptides, which can exploit changes in amino acid chirality to control metal ion coordination number and lead to an alternative path towards heterochromic systems. The constructs described herein represent the initial steps of preparing protein structures that may simultaneous contain structural and catalytic metal binding centers. These studies inform the community on a developing field, which promises new opportunities for the study of bioinorganic chemistry.

Metal Ion Substrate Inhibition of Ferrochelatase

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k(cat) and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) and can be alleviated by the addition of metal complexing agents such as beta-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.

Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases

Terminal RNA uridylyltransferases (TUTases) catalyze template-independent UMP addition to the 3' hydroxyl of RNA. TUTases belong to the DNA polymerase beta superfamily of nucleotidyltransferases that share a conserved catalytic domain bearing three metal-binding carboxylate residues. We have previously determined crystal structures of the UTP-bound and apo forms of the minimal trypanosomal TUTase, TbTUT4, which is composed solely of the N-terminal catalytic and C-terminal base-recognition domains. Here we report crystal structures of TbTUT4 with bound CTP, GTP, and ATP, demonstrating nearly perfect superposition of the triphosphate moieties with that of the UTP substrate. Consequently, at physiological nucleoside 5'-triphosphate concentrations, the protein-uracil base interactions alone are not sufficient to confer UTP selectivity. To resolve this ambiguity, we determined the crystal structure of a prereaction ternary complex composed of UTP, TbTUT4, and UMP, which mimics an RNA substrate, and the postreaction complex of TbTUT4 with UpU dinucleotide. The UMP pyrimidine ring stacks against the uracil base of the bound UTP, which on its other face also stacks with an essential tyrosine. In contrast, the different orientation of the purine bases observed in cocrystals with ATP and GTP prevents this triple stacking, precluding productive binding of the RNA. The 3' hydroxyl of the bound UMP is poised for in-line nucleophilic attack while contributing to the formation of a binding site for a second catalytic metal ion. We propose a dual role for RNA substrates in TUTase-catalyzed reactions: contribution to selective incorporation of the cognate nucleoside and shaping of the catalytic metal binding site.

The catalytic Mn2+ sites in the enolase-inhibitor complex: crystallography, single-crystal EPR, and DFT calculations.

Crystals of Zn2+/Mn2+ yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) were grown under conditions with a slight preference for binding of Zn2+ at the higher affinity site, site I. The structure of the Zn2+/Mn2+-PhAH complex was solved at a resolution of 1.54 A, and the two catalytic metal binding sites, I and II, show only subtle displacement compared to that of the corresponding complex with the native Mg2+ ions. Low-temperature echo-detected high-field (W-band, 95 GHz) EPR (electron paramagnetic resonance) and 1H ENDOR (electron-nuclear double resonance) were carried out on a single crystal, and rotation patterns were acquired in two perpendicular planes. Analysis of the rotation patterns resolved a total of six Mn2+ sites, four symmetry-related sites of one type and two out of the four of the other type. The observation of two chemically inequivalent Mn2+ sites shows that Mn2+ ions populate both sites I and II and the zero-field splitting (ZFS) tensors of the Mn2+ in the two sites were determined. The Mn2+ site with the larger D value was assigned to site I based on the 1H ENDOR spectra, which identified the relevant water ligands. This assignment is consistent with the seemingly larger deviation of site I from octahedral symmetry, compared to that of site II. The ENDOR results gave the coordinates of the protons of two water ligands, and adding them to the crystal structure revealed their involvement in a network of H bonds stabilizing the binding of the metal ions and PhAH. Although specific hyperfine interactions with the inhibitor were not determined, the spectroscopic properties of the Mn2+ in the two sites were consistent with the crystal structure. Density functional theory (DFT) calculations carried out on a cluster representing the catalytic site, with Mn2+ in site I and Zn2+ in site II, and vice versa, gave overestimated D values on the order of the experimental ones, although the larger D value was found for Mn2+ in site II rather than in site I. This discrepancy was attributed to the high sensitivity of the ZFS parameters to the Mn-O bond lengths and orientations, such that small, but significant, differences between the optimized and crystal structures alter the ZFS considerably, well above the difference between the two sites.

Global Methods to Monitor the Thiol–Disulfide State of ProteinsIn Vivo

Cysteines play an important role in protein biochemistry. The unique chemical property and high reactivity of the free thiol group makes reduced cysteine a versatile component of catalytic centers and metal binding sites in many cytosolic proteins and oxidized cystine a stabilizing component in many secreted proteins. Moreover, cysteines readily react with reactive oxygen and nitrogen species to form reversible oxidative thiol modifications. As a result, these reversible thiol modifications have found a use as regulatory nano-switches in an increasing number of redox sensitive proteins. These redox-regulated proteins are able to adjust their activity quickly in response to changes in their redox environment. Over the past few years, a number of techniques have been developed that give insight into the global thiol-disulfide state of proteins in the cell. They have been successfully used to find substrates of thiol-disulfide oxidoreductases and to discover novel redoxregulated proteins. This review will provide an overview of the current techniques, focus on approaches to quantitatively describe the extent of thiol modification in vivo, and summarize their applications.

Structural Insights into the Mechanism of Nuclease A, a ββα Metal Nuclease from Anabaena

Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and double-stranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the Δ(2-24),D121A mutant of NucA in the presence of Zn2+ and Mn2+ (PDB code 1ZM8). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a ββα metal finger motif and a hydrated Mn2+ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites ∼26 A apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a dimer. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg57 in Serratia, the structure determined here indicates that Arg93 in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the ββα metal finger nuclease family and available mutational data on NucA, we propose that His124 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state.

A pepD-like peptidase from the ruminal bacterium, Prevotella albensis

Peptidases of Prevotella spp. play an important role in the breakdown of protein to ammonia in the rumen. This study describes a peptidase cloned from Prevotella albensis M384. DNA from P. albensis was used to complement a peptidase-deficient strain of Escherichia coli, CM107. A cloned fragment, Pep581, which enabled growth of E. coli CM107, contained an ORF of 1452 bp, encoding a 484 amino acid residue protein with a calculated molecular weight of 53.2 kDa and a theoretical pI of 4.90. Pep581 shared similar sequence identity of 47% with PepD from E. coli, and it was also a metallo-aminopeptidase. A putative catalytic metal binding region was identified in Pep581, similar to that found in the related PepT (a tripeptidase) and PepA (an oligopeptidase). Gel filtration indicated Pep581 was a dimer in its native state, similar to PepD of E. coli. PepD is a broad specificity dipeptidase that has been found in several prokaryotes. The enzyme expressed from Pep581 differed from PepD enzymes previously characterised in that it hydrolysed tri- and oligopeptides in addition to dipeptides, cleaving single amino acids from the N terminus.