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1.
The [NiFe]-hydrogenase protein produced by many types of bacteria contains a dinuclear metal center that is required for enzymatic activity. Assembly of this metal cluster involves the coordinated activity of a number of helper proteins including the accessory protein, HypB, which is necessary for Ni(II) incorporation into the hydrogenase proteins. The HypB protein from Escherichia coli has two metal-binding sites, a high-affinity Ni(II) site that includes ligands from the N-terminal domain and a low-affinity metal site located within the C-terminal GTPase domain. In order to determine the physiological relevance of the two separate sites, hydrogenase production was assessed in strains of E. coli expressing wild-type HypB, the isolated GTPase domain, or site-directed mutants of metal-binding residues. These experiments demonstrate that both metal sites of HypB are critical for the maturation of the hydrogenase enzymes in E. coli. X-ray absorption spectroscopy of purified proteins was used to examine the detailed coordination spheres of each nickel-loaded site. In addition, because the low-affinity metal site has a stronger preference for Zn(II) than Ni(II), the ligands and geometry for this metal were also resolved. The results from these experiments are discussed in the context of a mechanism for Ni(II) insertion into the hydrogenase protein.  相似文献   

2.
Biosynthesis of the metallocenter in the active site of the [NiFe] hydrogenase enzyme requires the accessory protein HypB, which is a metal-binding GTPase. In this study, the interplay between the individual activities of Escherichia coli HypB was examined. The full-length protein undergoes nucleotide-responsive dimerization that is disrupted upon mutation of L242 and L246 to alanine. This mutant HypB is monomeric under all of the conditions investigated but the inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric protein has metal-binding behavior similar to that of wild-type HypB. Furthermore, expression of L242A/L246A HypB in vivo results in hydrogenase activity that is approximately half of the activity produced by the wild-type control, suggesting that dimerization of HypB does not have a critical role in the hydrogenase maturation pathway. In contrast, the GTPase activity of HypB is modulated by metal loading of the protein. These results provide insight into the role of HypB in hydrogenase biosynthesis.  相似文献   

3.
Leach MR  Sandal S  Sun H  Zamble DB 《Biochemistry》2005,44(36):12229-12238
The formation of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hydrogenase pleiotropy operon hypABCDEF. The insertion of Ni(II) into the precursor enzyme follows the incorporation of the iron center and is the function of HypA, a Zn(II)-binding protein, and HypB, a GTPase. The Ni(II) donor and the mechanism of transfer of Ni(II) into the hydrogenase precursor protein are not known. In this study, we demonstrate that HypB is a nickel-binding protein capable of binding 1 equiv of Ni(II) with a K(d) in the sub-picomolar range. In addition, HypB has a weaker metal-binding site that is not specific for Ni(II) over Zn(II). Examination of the isolated C-terminal GTPase domain revealed that the high-affinity metal binding capability was severely abrogated but the low-affinity site was intact. By mutating conserved cysteine and histidine residues in E. coli HypB, we have localized the high-affinity Ni(II)-binding site to an N-terminal CXXCGC motif and the low-affinity metal-binding site to the GTPase domain. A model for the function of HypB during the Ni(II) loading of hydrogenase is proposed.  相似文献   

4.
Chan KH  Li T  Wong CO  Wong KB 《PloS one》2012,7(1):e30547
Maturation of [NiFe]-hydrogenase requires the insertion of iron, cyanide and carbon monoxide, followed by nickel, to the catalytic core of the enzyme. Hydrogenase maturation factor HypB is a metal-binding GTPase that is essential for the nickel delivery to the hydrogenase. Here we report the crystal structure of Archeoglobus fulgidus HypB (AfHypB) in apo-form. We showed that AfHypB recognizes guanine nucleotide using Asp-194 on the G5 loop despite having a non-canonical NKxA G4-motif. Structural comparison with the GTPγS-bound Methanocaldococcus jannaschii HypB identifies conformational changes in the switch I region, which bring an invariant Asp-72 to form an intermolecular salt-bridge with another invariant residue Lys-148 upon GTP binding. Substitution of K148A abolished GTP-dependent dimerization of AfHypB, but had no significant effect on the guanine nucleotide binding and on the intrinsic GTPase activity. In vivo complementation study in Escherichia coli showed that the invariant lysine residue is required for in vivo maturation of hydrogenase. Taken together, our results suggest that GTP-dependent dimerization of HypB is essential for hydrogenase maturation. It is likely that a nickel ion is loaded to an extra metal binding site at the dimeric interface of GTP-bound HypB and transferred to the hydrogenase upon GTP hydrolysis.  相似文献   

5.
The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the k(cat) and K(m) of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes.  相似文献   

6.
CooC proteins are ATPases involved in the incorporation of nickel into the complex active site ([Ni-4Fe-4S]) cluster of Ni,Fe-dependent carbon monoxide dehydrogenases. The genome of the carboxydotrophic bacterium Carboxydothermus hydrogenoformans encodes five carbon monoxide dehydrogenases and three CooC-type proteins, of which CooC1 was shown to be a nickel-binding ATPase. We determined the crystal structure of CooC1 in four different states: empty, ADP-bound, Zn2+/ADP-bound, and Zn2+-bound. The structure of CooC1 consists of two spatially separated functional modules: an ATPase module containing the deviant Walker A motif and a metal-binding module that confers the specific function of CooC1. The ATPase module is homologous to other members of the MinD family and, in analogy to the dimeric structure of ATP-bound Soj, is likely responsible for the ATP-dependent dimerization of CooC1. Its core topology classifies CooC1 as a member of the MinD family of SIMIBI (signal recognition particle, MinD and BioD)-class NTPases. The crystal structure of Zn2+-bound CooC1 reveals a conserved C-X-C motif as the metal-binding site responsible for metal-induced dimerization. The competitive binding of Ni2+ and Zn2+ to CooC1 in solution confirms that the conserved C-X-C motif is also responsible for the interaction with Ni2+. A comparison of the different CooC1 structures determined suggests a mutual dependence of metal-binding site and nucleotide-binding site.  相似文献   

7.
The Escherichia coli protein SlyD is a member of the FK-506-binding protein family of peptidylprolyl isomerases. In addition to its peptidylprolyl isomerase domain, SlyD is composed of a molecular chaperone domain and a C-terminal tail rich in potential metal-binding residues. SlyD interacts with the [NiFe]-hydrogenase accessory protein HypB and contributes to nickel insertion during biosynthesis of the hydrogenase metallocenter. This study examines the HypB-SlyD complex and its significance in hydrogenase activation. Protein variants were prepared to delineate the interface between HypB and SlyD. Complex formation requires the HypB linker region located between the high affinity N-terminal Ni(II) site and the GTPase domain of the protein. In the case of SlyD, the deletion of a short loop in the chaperone domain abrogates the interaction with HypB. Mutations in either protein that disrupt complex formation in vitro also result in deficient hydrogenase production in vivo, indicating that the contact between HypB and SlyD is important for hydrogenase maturation. Surprisingly, SlyD stimulates release of nickel from the high affinity Ni(II)-binding site of HypB, an activity that is also disrupted by mutations that affect complex formation. Furthermore, a SlyD truncation lacking the C-terminal metal-binding tail still interacts with HypB but is deficient in stimulating metal release and is not functional in vivo. These results suggest that SlyD could activate metal release from HypB during metallation of the [NiFe] hydrogenase.  相似文献   

8.
The large subunit HoxC of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.  相似文献   

9.
The biosynthesis of [NiFe] hydrogenases is a complex process that requires the function of the Hyp proteins HypA, HypB, HypC, HypD, HypE, HypF, and HypX for assembly of the H(2)-activating [NiFe] site. In this study we examined the maturation of the regulatory hydrogenase (RH) of Ralstonia eutropha. The RH is a H(2)-sensing [NiFe] hydrogenase and is required as a constituent of a signal transduction chain for the expression of two energy-linked [NiFe] hydrogenases. Here we demonstrate that the RH regulatory activity was barely affected by mutations in hypA, hypB, hypC, and hypX and was not substantially diminished in hypD- and hypE-deficient strains. The lack of HypF, however, resulted in a 90% decrease of the RH regulatory activity. Fourier transform infrared spectroscopy and the incorporation of (63)Ni into the RH from overproducing cells revealed that the assembly of the [NiFe] active site is dependent on all Hyp functions, with the exception of HypX. We conclude that the entire Hyp apparatus (HypA, HypB, HypC, HypD, HypE, and HypF) is involved in an efficient incorporation of the [NiFe] center into the RH.  相似文献   

10.
The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and Pi, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination.  相似文献   

11.
HypA and HypB are maturation proteins required for incorporation of nickel into the hydrogenase large subunit. To examine the functions of these proteins in nickel insertion, the hybF gene, which is a homolog of hypA essential for maturation of hydrogenases 1 and 2 from Escherichia coli, was overexpressed, and the product was purified. This protein behaves like a monomer in gel filtration and contains stoichiometric amounts of zinc but insignificant or undetectable amounts of nickel and iron. In filter binding assays radioactively labeled nickel binds to HybF with a K(D) of 1.87 microM and in a stoichiometric ratio. To identify amino acid residues of HybF involved in nickel and/or zinc binding, variants in which conserved residues were replaced were studied. An H2Q replacement eliminated both in vivo activity and in vitro binding of nickel. The purified protein, however, contained zinc at the level characteristic of the wild-type protein. When E3 was replaced by Q, activity was retained, but an E3L exchange was detrimental. Replacement of each of the four conserved cysteine residues of a zinc finger motif reduced the cellular amount of HybF protein without a loss of in vivo activity, indicating that these residues play a purely structural role. A triple mutant deficient in the synthesis or activity of HypA, HybF, and HypB was constructed, and it exhibited the same responsiveness for phenotypic complementation by high nickel as mutants with a single lesion in one of the genes exhibited. The results are interpreted in terms of a concerted action of HypB and HybF in nickel insertion in which HybF (as well as its homolog, HypA) functions as a metallochaperone and HypB functions as a regulator that controls the interaction of HybF with the target protein.  相似文献   

12.
The maturation of [NiFe]-hydrogenase is highly dependent on a battery of chaperone proteins. Among these, HypA and HypB were proposed to exert nickel delivery functions in the metallocenter assembly process, although the detailed mechanism remains unclear. Herein, we have overexpressed and purified wild-type HypB as well as two mutants, K168A and M186L/F190V, from Helicobacter pylori. We demonstrated that all proteins bind Ni(2+) at a stoichiometry of one Ni(2+) per monomer of the proteins with dissociation constants at micromolar levels. Ni(2+) elevated GTPase activity of WT HypB, which is attributable to a lower affinity of the protein toward GDP as well as Ni(2+)-induced dimerization. The disruption of GTP-dependent dimerization has led to GTPase activities of both mutants in apo-forms almost completely abolished, compared with the wild-type protein. The GTPase activity is partially restored for HypB(M186L/F190V) mutant but not for HypB(K168A) mutant upon Ni(2+) binding. HypB forms a complex with its partner protein HypA with a low affinity (K(d) of 52.2 ± 8.8 μM). Such interactions were also observed in vivo both in the absence and presence of nickel using a GFP-fragment reassembly technique. The putative protein-protein interfaces on H. pylori HypA and HypB proteins were identified by NMR chemical shift perturbation and mutagenesis studies, respectively. Intriguingly, the unique N terminus of H. pylori HypB was identified to participate in the interaction with H. pylori HypA. These structural and functional studies provide insight into the molecular mechanism of Ni(2+) delivery during maturation of [NiFe]-hydrogenase.  相似文献   

13.
In the human gastric bacterium Helicobacter pylori, two metalloenzymes, hydrogenase and urease, are essential for in vivo colonization, the latter being a major virulence factor. The UreA and UreB structural subunits of urease and UreG, one of the accessory proteins for Ni(2+) incorporation into apourease, were taken as baits for tandem affinity purification. The method allows the purification of protein complexes under native conditions and physiological expression levels of the bait protein. Furthermore the tandem affinity purification technology was combined with in vivo cross-link to capture transient interactions. The results revealed different populations of urease complexes: (i) urease captured during activation by Ni(2+) ions comprising all the accessory proteins and (ii) urease in association with metabolic proteins involved e.g. in ammonium incorporation and the cytoskeleton. Using UreG as a bait protein, we copurified HypB, the accessory protein for Ni(2+) incorporation into hydrogenase, that is reported to play a role in urease activation. The interactome of HypB partially overlapped with that of urease and revealed interactions with SlyD, which is known to be involved in hydrogenase maturation as well as with proteins implicated in the formation of [Fe-S] clusters present in the small subunit of hydrogenase. In conclusion, this study provides new insight into coupling of ammonium production and assimilation in the gastric pathogen and the intimate link between urease and hydrogenase maturation.  相似文献   

14.
Chan KH  Lee KM  Wong KB 《PloS one》2012,7(2):e32592
The active site of [NiFe]-hydrogenase contains nickel and iron coordinated by cysteine residues, cyanide and carbon monoxide. Metal chaperone proteins HypA and HypB are required for the nickel insertion step of [NiFe]-hydrogenase maturation. How HypA and HypB work together to deliver nickel to the catalytic core remains elusive. Here we demonstrated that HypA and HypB from Archaeoglobus fulgidus form 1:1 heterodimer in solution and HypA does not interact with HypB dimer preloaded with GMPPNP and Ni. Based on the crystal structure of A. fulgidus HypB, mutants were designed to map the HypA binding site on HypB. Our results showed that two conserved residues, Tyr-4 and Leu-6, of A. fulgidus HypB are required for the interaction with HypA. Consistent with this observation, we demonstrated that the corresponding residues, Leu-78 and Val-80, located at the N-terminus of the GTPase domain of Escherichia coli HypB were required for HypA/HypB interaction. We further showed that L78A and V80A mutants of HypB failed to reactivate hydrogenase in an E. coli ΔhypB strain. Our results suggest that the formation of the HypA/HypB complex is essential to the maturation process of hydrogenase. The HypA binding site is in proximity to the metal binding site of HypB, suggesting that the HypA/HypB interaction may facilitate nickel transfer between the two proteins.  相似文献   

15.
The [NiFe] centers at the active sites of the Escherichia coli hydrogenase enzymes are assembled by a team of accessory proteins that includes the products of the hyp genes. To determine whether any other proteins are involved in this process, the sequential peptide affinity system was used. The analysis of the proteins in a complex with HypB revealed the peptidyl-prolyl cis/trans-isomerase SlyD, a metal-binding protein that has not been previously linked to the hydrogenase biosynthetic pathway. The association between HypB and SlyD was confirmed by chemical cross-linking of purified proteins. Deletion of the slyD gene resulted in a marked reduction of the hydrogenase activity in cell extracts prepared from anaerobic cultures, and an in-gel assay was used to demonstrate diminished activities of both hydrogenase 1 and 2. Western analysis revealed a decrease in the final proteolytic processing of the hydrogenase 3 HycE protein, indicating that the metal center was not assembled properly. These deficiencies were all rescued by growth in medium containing excess nickel, but zinc did not have any phenotypic effect. Experiments with radioactive nickel demonstrated that less nickel accumulated in DeltaslyD cells compared with wild type, and overexpression of SlyD from an inducible promoter doubled the level of cellular nickel. These experiments demonstrate that SlyD has a role in the nickel insertion step of the hydrogenase maturation pathway, and the possible functions of SlyD are discussed.  相似文献   

16.
The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX2C motifs characteristic of metal-binding proteins. In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX2CX81CX2C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.  相似文献   

17.
DNA protection during starvation (Dps) proteins play an important role in protecting cellular macromolecules from damage by reactive oxygen species (ROS). Unlike most orthologs that protect DNA by a combination of DNA binding and prevention of hydroxyl radical formation by ferroxidation and sequestration of iron, Dps-1 from the radiation-resistant Deinococcus radiodurans fails to protect DNA from hydroxyl radical-mediated cleavage through a mechanism inferred to involve continuous release of iron from the protein core. To address the structural basis for this unusual release of Fe(2+), the crystal structure of D. radiodurans Dps-1 was determined to 2.0 Angstroms resolution. Two of four strong anomalous signals per protein subunit correspond to metal-binding sites within an iron-uptake channel and a ferroxidase site, common features related to the canonical functions of Dps homologs. Similar to Lactobacillus lactis Dps, a metal-binding site is found at the N-terminal region. Unlike other metal sites, this site is located at the base of an N-terminal coil on the outer surface of the dodecameric protein sphere and does not involve symmetric association of protein subunits. Intriguingly, a unique channel-like structure is seen featuring a fourth metal coordination site that results from 3-fold symmetrical association of protein subunits through alpha2 helices. The presence of this metal-binding site suggests that it may define an iron-exit channel responsible for the continuous release of iron from the protein core. This interpretation is supported by substitution of residues involved in this ion coordination and the observation that the resultant mutant protein exhibits significantly attenuated iron release. Therefore, we propose that D. radiodurans Dps-1 has a distinct iron-exit channel.  相似文献   

18.
Lindner HA  Alary A  Boju LI  Sulea T  Ménard R 《Biochemistry》2005,44(48):15645-15651
The aminoacylase-1/metallopeptidase 20 (Acy1/M20) family is the largest metallopeptidase family. Several crystal structures feature a metal-binding and a dimerization-mediating domain, both arranged in an extended open conformation. We have recently shown [Lindner et al. (2003) J. Biol. Chem. 278, 44496-44504] that in human Acy1 the invariant residues Glu147 and His206 from the metal-binding and the dimerization domain, respectively, are recruited to the active site from opposite dimer subunits. We hypothesized that, to facilitate this, formation of the binary complex is associated with domain closure, which would also position additional residues in the functional active site of Acy1. These would include two partially conserved dimerization domain residues: an asparagine (Asn263) and an arginine (Arg276) from the same subunit as His206 and Glu147, respectively. In this paper, we investigate the significance of the three dimerization domain residues of human Acy1 His206, Asn263, and Arg276 and, additionally, the nearby Asp274 for catalysis using site-directed mutagenesis. Enzyme complementation assays confirm the putative subunit allocations of these residues, and steady-state kinetics support roles for all of them in catalysis but only involve the Arg276 in substrate-binding. The results are consistent with a model of the closed conformation for the structure of the related enzyme carboxypeptidase G2. This study demonstrates experimentally for the first time for a member of the Acy1/M20 family that several residues outside of the metal-binding domain are involved in binding and catalysis.  相似文献   

19.
Sun X  Xiao CL  Ge R  Yin X  Li H  Li N  Yang X  Zhu Y  He X  He QY 《Proteomics》2011,11(16):3288-3298
The aim of metalloproteomics is to identify and characterize putative metal-binding proteins and metal-binding motifs. In this study, we performed a systematical metalloproteomic analysis on Streptococcus pneumoniae through the combined use of efficient immobilized metal affinity chromatography enrichment and high-accuracy linear ion trap-Orbitrap MS to identify metal-binding proteins and metal-binding peptides. In total, 232 and 166 putative metal-binding proteins were respectively isolated by Cu- and Zn-immobilized metal affinity chromatography columns, in which 133 proteins were present in both preparations. The putative metalloproteins are mainly involved in protein, nucleotide and carbon metabolisms, oxidation and cell cycle regulation. Based on the sequence of the putative Cu- and Zn-binding peptides, putative Cu-binding motifs were identified: H(X)mH (m=0-11), C(X)(2) C, C(X)nH (n=2-4, 6, 9), H(X)iM (i=0-10) and M(X)tM (t=8 or 12), while putative Zn-binding motifs were identified as follows: H(X)mH (m=1-12), H(X)iM (i=0-12), M(X)tM (t=0, 3 and 4), C(X)nH (n=1, 2, 7, 10 and 11). Equilibrium dialysis and inductively coupled plasma-MS experiments confirmed that the artificially synthesized peptides harboring differential identified metal-binding motifs interacted directly with the metal ions. The metalloproteomic study presented here suggests that the comparably large size and diverse functions of the S. pneumoniae metalloproteome may play important roles in various biological processes and thus contribute to the bacterial pathologies.  相似文献   

20.
Escherichia coli and related bacteria require nickel for the synthesis of hydrogenases, enzymes involved in hydrogen oxidation and proton reduction. Nickel transport to the cytoplasm depends on five proteins, NikA-E. We have previously reported the three-dimensional structure of the soluble periplasmic nickel transporter NikA in a complex with FeEDTA(H 2O) (-). We have now determined the structure of EDTA-free NikA and have found that it binds a small organic molecule that contributes three ligands to the coordination of a transition metal ion. Unexpectedly, His416, which was far from the metal-binding site in the FeEDTA(H 2O) (-)-NikA complex, becomes the fourth observed ligand to the metal. The best match to the omit map electron density is obtained for butane-1,2,4-tricarboxylate (BTC). Our attempts to obtain a BTC-Ni-NikA complex using apo protein and commercial reagents resulted in nickel-free BTC-NikA. Overall, our results suggest that nickel transport in vivo requires a specific metallophore that may be BTC.  相似文献   

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