Histidine-rich Region Research Articles

Deletion of a Histidine-rich Loop of AtMTP1, a Vacuolar Zn2+/H+ Antiporter of Arabidopsis thaliana, Stimulates the Transport Activity

Arabidopsis thaliana AtMTP1 belongs to the cation diffusion facilitator family and is localized on the vacuolar membrane. We investigated the enzymatic kinetics of AtMTP1 by a heterologous expression system in the yeast Saccharomyces cerevisiae, which lacked genes for vacuolar membrane zinc transporters ZRC1 and COT1. The yeast mutant expressing AtMTP1 heterologously was tolerant to 10 mm ZnCl(2). Active transport of zinc into vacuoles of living yeast cells expressing AtMTP1 was confirmed by the fluorescent zinc indicator FuraZin-1. Zinc transport was quantitatively analyzed by using vacuolar membrane vesicles prepared from AtMTP1-expressing yeast cells and radioisotope (65)Zn(2+). Active zinc uptake depended on a pH gradient generated by endogenous vacuolar H(+)-ATPase. The activity was inhibited by bafilomycin A(1), an inhibitor of the H(+)-ATPase. The K(m) for Zn(2+) and V(max) of AtMTP1 were determined to be 0.30 microm and 1.22 nmol/min/mg, respectively. We prepared a mutant AtMTP1 that lacked the major part (32 residues from 185 to 216) of a long histidine-rich hydrophilic loop in the central part of AtMTP1. Yeast cells expressing the mutant became hyperresistant to high concentrations of Zn(2+) and resistant to Co(2+). The K(m) and V(max) values were increased 2-11-fold. These results indicate that AtMTP1 functions as a Zn(2+)/H(+) antiporter in vacuoles and that a histidine-rich region is not essential for zinc transport. We propose that a histidine-rich loop functions as a buffering pocket of Zn(2+) and a sensor of the zinc level at the cytoplasmic surface. This loop may be involved in the maintenance of the level of cytoplasmic Zn(2+).

Histidine-Rich Glycoprotein Modulation of Immune/Autoimmune, Vascular, and Coagulation Systems

Histidine-rich glycoprotein (HRG) is plasma glycoprotein that has a multidomain structure, interacts with many ligands, and exhibit many modulatory functions in diverse biological systems. HRG ligands include Zn2+, tropomyosin, heparin and heparan sulphate, plasminogen, plasmin, fibrinogen, thrombospondin, IgG, FcgR, and complement. In many cases, the histidine-rich region of the molecule enhances ligand binding after interaction with Zn2+ or exposure to low pH, conditions associated with sites of tissue injury or tumor growth. The multidomain nature of HRG and diverse ligand binding properties indicates that it can act as an extracellular adaptor protein, bridging together different ligands mainly on cell surfaces. Apart from cell surface molecules, HRG can differentially target IgG, preventing generation of insoluble immune complexes. HRG binds to most cells primarily via heparan sulphate proteoglycans. HRG can enhance clearance of apoptotic and necrotic phagocytes as well as immune complexes. The anti-angiogenic properties of HRG are well established in vitro and in vivo. HRG can modulate other physiological processes such as cell adhesion and migration, fibrinolysis and coagulation, complement activation. This review presents an update on the molecular, structural, biological, and clinical properties of HRG and associated autoimmunity conditions.

Cloning and functional identification of two members of the ZIP (Zrt, Irt-like protein) gene family in rice (Oryza sativa L.)

Two ZIP (Zrt, Irt-like Protein) cDNAs were isolated from rice (Oryza sativa L.) by RT-PCR approach, and named as OsZIP7a and OsZIP8 respectively. The predicted proteins of OsZIP7a and OsZIP8 consist of 384 and 390 amino acid residues respectively, and display high similarity to other plant ZIP proteins. Each protein contains eight transmembrane (TM) domains and a highly conserved ZIP signature motif, with a histidine-rich region in the variable region between TM domains III and IV. By semi-quantitative RT-PCR approach, it was found that the expression of OsZIP7a was significantly induced in rice roots by iron-deficiency, while that of OsZIP8 induced in both rice roots and shoots by zinc-deficiency. When expressed in yeast cells, OsZIP7a and OsZIP8 could complement an iron-uptake-deficient yeast mutant and a zinc-uptake-deficient yeast mutant respectively. It suggested that the OsZIP7a and OsZIP8 might encode an iron and a zinc transporter protein in rice respectively.

A Nonclassical Arabinogalactan Protein Gene Highly Expressed in Vascular Tissues, AGP31, Is Transcriptionally Repressed by Methyl Jasmonic Acid in Arabidopsis

In response to wounding and pathogens, jasmonate (JA) serves as a signal molecule for both induction and repression of gene expression. To examine defense-regulated gene repression in Arabidopsis (Arabidopsis thaliana), we have identified a nonclassical arabinogalactan protein (AGP) gene, AGP31, and show that its mRNA decreased to about 30% of its original level within 8 h in response to methyl JA (MeJA) treatment of whole 7-d-old seedlings. Wounding and abscisic acid treatment had similar effects. MeJA suppression primarily depends on the action of the JA-signaling protein, COI1, as shown by much lower MeJA suppression in coi1-1 mutant plants. The main mechanism of mRNA suppression by MeJA is repression of transcription, as shown by nuclear run-on experiments. The AGP31 protein shares features with several known and putative nonclassical AGPs from other species: a putative signal peptide, a histidine-rich region near the N terminus followed by a repetitive proline-rich domain, and a cysteine-rich C-terminal PAC (for proline-rich protein and AGP, containing cysteine) domain. Positive Yariv reagent interaction demonstrated that the protein is an AGP. Monosaccharide analysis of purified AGP31 indicated it is a galactose-rich AGP. Expression of an AGP31-enhanced green fluorescent protein fusion protein in transgenic cells revealed that the AGP31 protein was localized to the cell wall. AGP31 promoter-beta-glucuronidase reporter gene analysis showed expression in the vascular bundle throughout the plant, except in the flower. In the flower, beta-glucuronidase staining occurred throughout the pistil, except in the stigma. The strong preferential expression in vascular tissues suggests that AGP31 may be involved in vascular tissue function during both the defense response and development.

Four domains of p300 each bind tightly to a sequence spanning both transactivation subdomains of p53

The transcriptional coactivator p300 binds to and mediates the transcriptional functions of the tetrameric tumor suppressor p53. Both proteins consist of independently folded domains linked by intrinsically disordered sequences. A well studied short sequence of the p53 transactivation domain, p53(15-29), binds weakly to four folded domains of p300 [Taz1/cysteine-histidine-rich region 1 (CH1), Kix, Taz2/CH3, IBiD], with dissociation constants (K(D)) in the 100 muM region. However, we found that a longer N-terminal transactivation domain construct p53(1-57) bound tightly to each p300 domain. Taz2/CH3 had the greatest affinity (K(D) = 27 nM) and competes with the N-terminal domain of Mdm2 for the p53 N terminus. p300 thus can protect the N terminus of p53 against the binding of other proteins. Mutations of p53 that abrogate transactivation (L22Q/W23S, W53Q/F54S) greatly weakened binding to each p300 domain, linking phenotypic defects to weakened coactivator binding. We propose a complex between tetrameric p53 and p300 in which four domains of p300 wrap around the four transactivation domains of p53.

Histidine‐rich glycoprotein exerts antibacterial activity

Histidine-rich glycoprotein (HRGP), an abundant heparin-binding protein found in plasma and thrombocytes, exerts antibacterial effects against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Fluorescence studies and electron microscopy to assess membrane permeation showed that HRGP induces lysis of E. faecalisbacteria in the presence of Zn2+ or at low pH. Heparin blocked binding of the protein to E. faecalis and abolished antibacterial activity. Furthermore, truncated HRGP, devoid of the heparin-binding and histidine-rich domain, was not antibacterial. It has previously been shown that peptides containing consensus heparin-binding sequences (Cardin and Weintraub motifs) are antibacterial. Thus, the peptide (GHHPH)4, derived from the histidine-rich region of HRGP and containing such a heparin-binding motif, was antibacterial for E. faecalis in the presence of Zn2+ or at low pH. The results show a previously undisclosed antibacterial activity of HRGP and suggest that the histidine-rich and heparin-binding domain of HRGP mediates the antibacterial activity of the protein.

Structural Insights into HypB, a GTP-binding Protein That Regulates Metal Binding

HypB is a prokaryotic metal-binding guanine nucleotide-binding protein that is essential for nickel incorporation into hydrogenases. Here we solved the x-ray structure of HypB from Methanocaldococcus jannaschii. It shows that the G-domain has a different topology than the Ras-like proteins and belongs to the SIMIBI (after Signal Recognition Particle, MinD and BioD) class of NTP-binding proteins. We show that HypB undergoes nucleotide-dependent dimerization, which is apparently a common feature of SIMIBI class G-proteins. The nucleotides are located in the dimer interface and are contacted by both subunits. The active site features residues from both subunits arguing that hydrolysis also requires dimerization. Two metal-binding sites are found, one of which is dependent on the state of bound nucleotide. A totally conserved ENV/IGNLV/ICP motif in switch II relays the nucleotide binding with the metal ionbinding site. The homology with NifH, the Fe protein subunit of nitrogenase, suggests a mechanistic model for the switch-dependent incorporation of a metal ion into hydrogenases.

Unexpected diversity displayed in cDNAs expressed by the immune cells of the purple sea urchin,Strongylocentrotus purpuratus

We recently identified a unique family of transcripts, the 185/333 family, that comprise approximately 60% of the mRNAs induced by coelomocytes from the purple sea urchin in response to immunological challenge from lipopolysaccharide. An analysis of 81 full-length cDNAs revealed 67 unique nucleotide sequences encoding 64 different proteins. Diversity of the transcripts was based on 25 sequence blocks, or "elements," which resulted in 22 different element patterns based on their presence or absence. Furthermore, there was a high level of nucleotide variation within elements, including single nucleotide polymorphisms and insertions/deletions, both of which resulted in amino acid sequence variability. The deduced 185/333 proteins contained an NH2-terminal leader, a glycine-rich region with an RGD motif, a histidine-rich region, and a COOH-terminal region. Two 185/333 genes, identified in the partially assembled Strongylocentrotus purpuratus genome, have two exons. The first encoded the leader, and the second encoded the remainder of the predicted protein. Estimates from quantitative PCR indicated that there were approximately 100 alleles in the diploid genome. These results suggested that the purple sea urchin may have mechanisms for generating high levels of diversity in response to immunological challenge that have not been considered previously.

The cysteine–histidine-rich region of the movement protein of Cucumber mosaic virus contributes to plasmodesmal targeting, zinc binding and pathogenesis

Viral movement proteins (MPs) are central to the establishment of viral pathogenesis, and yet relatively little is understood about the structural and functional aspects of MPs or about the host factors on which they depend. Through chemical mutagenesis of transgenic Arabidopsis expressing Cucumber mosaic virus (CMV) MP fused with the green fluorescent protein, we have studied the function of a central region of the MP, defined by a number of conserved cysteine and histidine residues (Cys–His-rich region), which potentially functions as a zinc-binding domain. Transient expression of mutant MPs identified through an in planta screen for altered MP function or constructed with altered putative zinc ligands through site-directed mutagenesis showed that mutations in the Cys–His-rich region affected localization to and trafficking through plasmodesmata. In vitro zinc-binding analysis revealed that wild type (wt) CMV MP had the ability to bind zinc and that movement-defective mutants bound zinc with less affinity than wt MP. Furthermore, a correlation between the association of the MP with plasmodesmata and virus pathogenesis was shown. We discuss roles of the Cys–His region in biochemical and biological functions of the MP during virus movement.

X-ray Absorption Investigation of a Unique Protein Domain Able To Bind both Copper(I) and Copper(II) at Adjacent Sites of the N-Terminus of Haemophilus ducreyi Cu,Zn Superoxide Dismutase

The N-terminal metal binding extension of the Cu,Zn superoxide dismutase from Haemophilus ducreyi is constituted by a histidine-rich region followed by a methione-rich sequence which shows high similarity with protein motifs involved in the binding of Cu(I). X-ray absorption spectroscopy experiments selectively carried out with peptides corresponding to the two metal binding regions indicate that both sequences can bind either Cu(II) or Cu(I). However, competition experiments demonstrate that Cu(II) is preferred by histidine residues belonging to the first half of the motif, while the methionine-rich region preferentially binds Cu(I) via the interaction with three methionine sulfur atoms. Moreover, we have observed that the rate of copper transfer from the peptides to the active site of a copper-free form of the Cu,Zn superoxide dismutase mutant lacking the N-terminal extension depends on the copper oxidation state and on the residues involved in metal binding, histidine residues being critically important for the efficient transfer. Differences in the enzyme reactivation rates in the presence of mixtures of the two peptides when compared to those obtained with the single peptides suggest that the two halves of the N-terminal domain functionally interact during the process of copper transfer, possibly through subtle modifications of the copper coordination environment.

Histidine‐rich glycoprotein: A novel adaptor protein in plasma that modulates the immune, vascular and coagulation systems

Histidine-rich glycoprotein (HRG) is an abundant plasma glycoprotein that has a multidomain structure, interacts with many ligands, and has been shown to regulate a number of important biological processes. HRG ligands include Zn(2+) and haem, tropomyosin, heparin and heparan sulphate, plasminogen, plasmin, fibrinogen, thrombospondin, IgG, FcgammaR and complement. In many cases, the histidine-rich region of the molecule enhances ligand binding following interaction with Zn(2+) or exposure to low pH, conditions associated with sites of tissue injury or tumour growth. The multidomain nature of HRG indicates that it can act as an extracellular adaptor protein, bringing together disparate ligands, particularly on cell surfaces. HRG binds to most cells primarily via heparan sulphate proteoglycans, binding which is also potentiated by elevated free Zn(2+) levels and low pH. Recent reports have shown that HRG can modulate angiogenesis and additional studies have shown that it may regulate other physiological processes such as cell adhesion and migration, fibrinolysis and coagulation, complement activation, immune complex clearance and phagocytosis of apoptotic cells. This review outlines the molecular, structural, biological and clinical properties of HRG as well as describing the role of HRG in various physiological processes.

Granulin and Granulin Repeats Interact with the Tat·P-TEFb Complex and Inhibit Tat Transactivation

The cellular positive transcription elongation factor b (P-TEFb), containing cyclin T1 and cyclin-dependent kinase 9 (CDK9), interacts with the human immunodeficiency virus, type 1 (HIV-1) regulatory protein Tat to enable viral transcription and replication. Cyclin T1 is an unusually long cyclin and is engaged by cellular regulatory proteins. Previous studies showed that the granulin/epithelin precursor (GEP) binds the histidine-rich region of cyclin T1 and inhibits P-TEFb function. GEP is composed of repeats that vary in sequence and properties. GEP also binds directly to Tat. Here we show that GEP and some of its constituent granulin repeats can inhibit HIV-1 transcription via Tat without directly binding to cyclin T1. The interactions of granulins with Tat and cyclin T1 differ with respect to their binding sites and divalent cation requirements, and we identified granulin repeats that bind differentially to Tat and cyclin T1. Granulins DE and E bind Tat but do not interact directly with cyclin T1. These granulins are present in complexes with Tat and P-TEFb in which Tat forms a bridge between the cellular proteins. Granulins DE and E repress transcription from the HIV-1 LTR and gene expression from the viral genome, raising the possibility of developing granulin-based inhibitors of viral infection.

A Multifunctional Lipoxygenase with Fatty Acid Hydroperoxide Cleaving Activity from the Moss Physcomitrella patens

A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.

Primary structures of guinea pig high- and low-molecular-weight kininogens

Guinea pig high-molecular-weight and low-molecular-weight (HMW and LMW) kininogen cDNA were amplified from liver mRNA by RT-PCR. Their nucleotide sequences were analyzed and deduced to amino acid sequences. The HMW kininogen, composed of 607 amino acid residues with a 18-residue signal sequence, possessed the cysteine protease inhibitor domains I and II, the bradykinin domain, the histidine-rich region, and the prekallikrein-binding region. The amino acid sequence preceding the bradykinin domain was found not to be –Leu–Met–Lys– but –Leu–Thr–Arg–. Therefore, kallidin (Lys-bradykinin) and Met-kallidin are not liberated from the guinea pig kininogens. We purified the HMW kininogen protein from plasma and prepared the kinin-free form using guinea pig plasma kallikrein. Although the amino-terminal of the HMW kininogen was modified, the 25 amino-terminal residues of the light chain of the kinin-free kininogen corresponded to the deduced sequence just after the bradykinin moiety of the HMW kininogen. With regard to the LMW kininogen, the nucleotide sequence down to T1200 as well as the amino acid sequence till Thr382 was identical to that of the HMW kininogen. We also examined the localization of the guinea pig kininogen gene on the prometaphase lymphocyte chromosomes by fluorescence in situ hybridization method. Two pair signals were observed on a pair of homologous chromosomes, each of which is composed of two chromatids. Based on these findings, we concluded that HMW and LMW kininogens are produced from the single kininogen gene in guinea pig as in the cases of the other mammalian species reported so far.

Histidine-rich Glycoprotein Binds to Cell-surface Heparan Sulfate via Its N-terminal Domain following Zn2+ Chelation

Histidine-rich glycoprotein (HRG) is an alpha2-glycoprotein found in mammalian plasma at high concentrations (approximately 150 microg/ml) and is distinguished by its high content of histidine and proline. Structurally, HRG is a modular protein consisting of an N-terminal cystatin-like domain (N1N2), a central histidine-rich region (HRR) flanked by proline-rich sequences, and a C-terminal domain. HRG binds to cell surfaces and numerous ligands such as plasminogen, fibrinogen, thrombospondin, C1q, heparin, and IgG, suggesting that it may act as an adaptor protein either by targeting ligands to cell surfaces or by cross-linking soluble ligands. Despite the suggested functional importance of HRG, the cell-binding characteristics of the molecule are poorly defined. In this study, HRG was shown to bind to most cell lines in a Zn(2+)-dependent manner, but failed to interact with the Chinese hamster ovary cell line pgsA-745, which lacks cell-surface glycosaminoglycans (GAGs). Subsequent treatment of GAG-positive Chinese hamster ovary cells with mammalian heparanase or bacterial heparinase III, but not chondroitinase ABC, abolished HRG binding. Furthermore, blocking studies with various GAG species indicated that only heparin was a potent inhibitor of HRG binding. These data suggest that heparan sulfate is the predominate cell-surface ligand for HRG and that mammalian heparanase is a potential regulator of HRG binding. Using recombinant forms of full-length HRG and the N-terminal N1N2 domain, it was shown that the N1N2 domain bound specifically to immobilized heparin and cell-surface heparan sulfate. In contrast, synthetic peptides corresponding to the Zn(2+)-binding HRR of HRG did not interact with cells. Furthermore, the binding of full-length HRG, but not the N1N2 domain, was greatly potentiated by physiological concentrations of Zn2+. Based on these data, we propose that the N1N2 domain binds to cell-surface heparan sulfate and that the interaction of Zn2+ with the HRR can indirectly enhance cell-surface binding.

Three fur homologues from Anabaena sp. PCC7120: exploring reciprocal protein-promoter recognition

DNA sequence analysis of the Anabaena sp. PCC7120 genome confirmed the presence of three open reading frames ( all1691, all2473 and alr0957) containing the histidine-rich region characteristic of the Fur family. The genes coding for the three Fur proteins were cloned and overexpressed in Escherichia coli. The overexpression products, called FurA, FurB and FurC are only distantly related. The ability of the three recombinant proteins to bind iron-boxes identified in the three fur promoter regions was tested by electrophoretical mobility shift assays. FurA binds the three fur promoters with increased affinity in presence of metal. FurB also binds the three fur promoters, and its affinity is increased with DTT. FurC does not bind to furA or furB promoter regions or to its own promoter. However, FurC affects the ability of FurB and FurA to bind their target promoters.

Isolation, characterization and expression of the hex1 gene from Trichoderma reesei

Polymers of the HEX1 protein produce Woronin bodies in filamentous fungi. We have isolated and sequenced the hex1 gene and flanking regions from the industrially exploited fungus Trichoderma reesei. Multiple transcription start sites (TSS) and the 5' untranslated region (UTR) were identified by 5'RACE PCR. There are three hex1 transcript types, two of which originate from two TSSs at approximately -320 and -1335 from the start codon, which are separated by a 500-bp intron within the 5'UTR. The third transcript type results from alternative splicing of the intron within the coding sequence at the 3' end, which results in the inclusion or exclusion of an unconserved histidine-rich coding region. The three transcripts code for two forms of HEX1 protein. N-terminal sequencing of HEX1 separated by 2D gel electrophoresis confirms that there are two forms of HEX1 protein which are modified further by alternative cleavage of the N-terminus. The dominant form of HEX1 is coded by a cDNA with TSS at position -1335. Expression of hex1 on cellulase-inducing medium peaks strongly within 24 h of growth but the protein is expressed at a lower and more consistent level in medium containing glucose. This is the first investigation of expression of the hex1 gene encoding a protein unique to filamentous fungi.

Identification and characterization of several new members of the ZIP family of metal ion transporters in Medicago truncatula.

To broaden our understanding of micronutrient metal transport in plants, we have identified cDNAs for six new metal transporters in the model legume Medicago truncatula. All of the predicted proteins have high similarity to the ZIP protein family, and have been designated MtZIP1, MtZIP3, MtZIP4, MtZIP5, MtZIP6, and MtZIP7. The six predicted proteins ranged from 350 to 372 amino acids in length; sequence analysis revealed that all proteins contained eight transmembrane domains and the highly conserved ZIP signature motif. Most of the proteins also exhibited a histidine-rich region in the variable sequence between transmembrane domains III and IV. When MtZIPs were transformed into appropriate metal-uptake defective yeast mutants and grown on metal-limited media, MtZIP1, MtZIP5, and MtZIP6 proteins restored yeast growth on Zn-limited media, MtZIP4 and MtZIP7 proteins restored yeast growth on Mn-limited media, and MtZIP3, MtZIP5, and MtZIP6 proteins restored yeast growth on Fe-limited media. Therefore, we conclude that these proteins function as metal transporters in Medicago truncatula. The expression pattern for each gene was studied by semi-quantitative RT-PCR in roots and leaves from plants grown under various metal supplies. MtZIP1 transcripts were only detected in Zn-deficient roots and leaves. MtZIP3 and MtZIP4 expression was down regulated in leaves from Mn- and Fe-deficient plants and appeared to be upregulated under Zn-deficient conditions in both roots and leaves. MtZIP5 was upregulated in leaves under Zn and Mn deficiency. The expression of MtZIP6 and MtZIP7 was unaffected by the metal supply, at least in root and leaf tissues. Characterizing these proteins in a single organism will allow us to understand the interplay between various ZIP genes, and the role they play in the regulation/execution of plant metal homeostasis.

Structure–function studies of yeast C-4 sphingolipid long chain base hydroxylase

The roles of putative active site residues of the Saccharomyces cerevisiae sphingolipid C-4 long chain base hydroxylase (Sur2p) were investigated by site-directed mutagenesis. The replacement of any one of conserved His residues of three histidine-rich motifs with an alanine eliminated hydroxylase activity in vivo and in vitro, indicating that they are all essential elements of the active site. An additional conserved His residue (His 249) outside of the histidine-rich cluster region was also found to be crucial for activity. Additional mutants altered in residues in close proximity to the histidine-rich cluster were generated. In order to determine their roles in hydroxylase vs. desaturase activities, residues were replaced with conserved residues from the yeast Δ7-sterol-C5(6)-desaturase, Erg3p. Residues Phe 174, Asn 182, Ser 191, Leu 196, Pro 199, Asn 266, Tyr 269, Asp 271 and Gln 275 appear to be additionally important elements of the active site but their conversion into corresponding Erg3p residues did not lead to a gain in desaturase activity. It is concluded that Sur2p is a membrane-bound hydroxylase that belongs to the diiron family of eight-histidine motif enzymes.

Characterization of the Cobaltochelatase CbiXL: EVIDENCE FOR A 4Fe-4S CENTER HOUSED WITHIN AN MXCXXC MOTIF

CbiX is a cobaltochelatase required for the biosynthesis of vitamin B12 and is found in Archaea as a short form (CbiXS containing 120-145 amino acids) and in some bacteria as a longer version (CbiXL containing 300-350 amino acids). Purification of either recombinant Bacillus megaterium or Synechocystis CbiXL in Escherichia coli, which is facilitated by the presence of a naturally occurring histidine-rich region of the protein, results in the isolation of a dark brown protein solution. The UV/visible spectrum of the protein is consistent with the presence of a redox group, and the lack of definition within the spectrum is suggestive of a 4Fe-4S center. The presence of an iron-sulfur center was confirmed by EPR analysis of the proteins, which produces a pseudoaxial spectrum with g values at 2.04, 1.94, and 1.90. The EPR spectrum was absent at 70 K, an observation that is diagnostic of a 4Fe-4S center. Redox potentiometry coupled with optical spectroscopy allowed the midpoint potential of the redox center to be determined for the CbiXL from both B. megaterium and Synechocystis. Sequence analysis of CbiXL proteins reveals only two conserved cysteine residues within the CbiXL proteins, which are part of an MXCXXC motif. Mutagenesis of the two cysteines leads to loss of both the EPR spectrum and UV/visible spectral features of the Fe-S center in the protein, clearly indicating that these residues are involved in ligating the cofactor to the apoprotein possibly in a butterfly arrangement. The potential physiological role of the iron-sulfur center is discussed.