Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein.

The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.     

Isolation of four novel tachykinins from frog (Rana catesbeiana) brain and intestine

In a survey for unknown bioactive peptides in frog (Rana catesbeiana) brain and intestine, we isolated four novel peptides that exhibit potent stimulant effects on smooth muscle preparation of guinea pig ileum. By microsequencing and synthesis, these peptides were identified as Lys- Pro- Ser- Pro- Asp- Arg- Phe- Tyr- Gly- Leu- Met- NH2 (ranatachykinin A), Tyr- Lys- Ser- Asp- Ser- Phe- Tyr- Gly- Leu- Met- NH2 (ranatachykinin B), His- Asn- Pro- Ala- Ser- Phe- Ile- Gly- Leu- Met- NH2 (ranatachykinin C) and Lys- Pro- Ans- Pro- Glu- Arg- Phe- Tyr- Ala- Pro- Met- NH2 (ranatachykinin D). Ranatachykinin (RTK) A, B and C conserve the C- terminal sequence, Phe- X- Gly- Leu- Met- NH2, which is common to known members of the tachykinin family. On the other hand, RTK-D has a striking feature in its C-terminal sequence, Phe- Tyr- Ala- Pro- Met- NH2, which has never been found in other known tachykinins, and may constitute a new subclass in the tachykinin family.     

Isolation and characterization of lactose permease mutants with an enhanced recognition of maltose and diminished recognition of cellobiose

In the present study, lactose permease mutants were isolated which have an enhanced recognition toward maltose (an alpha-glucoside) and diminished recognition for cellobiose (a beta-glucoside). Nine mutants were isolated from a strain encoding a wild-type permease (pTE18) and nine from a strain encoding a mutant permease which recognizes maltose (pB15). All 18 mutants were subjected to DNA sequencing, and it was found that all mutations are single base substitutions within the lac Y gene effecting single amino acid substitutions within the protein. From the pTE18 parent, substitutions involved Tyr-236 to Phe or His; Ser-306 to Thr; and six independent mutants in which Ala-389 was changed to Pro. From pB15, Tyr-236 was changed to Phe or Asn, Ser-306 to Thr or Leu, Lys-319 to Asn, and His-322 to Tyr, Asn, or Gln. All 18 mutants exhibited enhanced recognition for maltose (compared with the pTE18 strain) and a diminished recognition for cellobiose. In addition, all mutants showed a diminished recognition toward beta-galactosides as well. The Phe-236, His-236, Leu-306, Asn-319, Tyr-322, Asn-322, and Gln-322 mutants were completely defective in the uphill accumulation of methyl-beta-D-thiogalactopyranoside whereas the Asn-236, Thr-306, and Pro-389 mutants could effectively accumulate methyl-beta-D-thiogalactopyranoside against a concentration gradient. The mutants obtained in this study, together with previous lactose permease mutants, tend to be found on transmembrane segments, and those which are on the same transmembrane segment are often found three or four amino acids away from each other. This pattern is consistent with a protein structure in which important amino acid side chains project from several transmembrane segments in such a way as to form a hydrophilic channel for the recognition and transport of H+ and galactosides. It is proposed that the mechanism for H+/lactose cotransport is consistent with a "flanking gate" model in which the protein contains a single recognition site for galactosides within the channel which is flanked on either side by gates.     

Dependence of Helicobacter pylori urease activity on the nickel-sequestering ability of the UreE accessory protein

The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in E. coli and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type H. pylori and in hypA and hypB mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between H. pylori urease activity and the nickel-sequestering ability of the UreE accessory protein.     

Molecular dynamics simulation and bioinformatics study on yeast aquaporin Aqy1 from Pichia pastoris

In the present study, an equilibrated system for the Aqy1 tetramer was developed, and molecular biophysics modeling showed that the Aqy1 channel was blocked by Tyr-31 in the N-terminus, which was also supported by the free energy profiles. However, bioinformatics analysis of the amino acid sequence of Aqy1 indicated this Tyr-31 is not conserved across all fungi. Analysis of the equilibrated structure showed that the central pore along the four-fold axis of the tetramers is formed with hydrophobic amino acid residues. In particular, Phe-90, Trp-198, and Phe-202 form the narrowest part of the pore. Therefore, water molecules are not expected to translocate through the central pore, a hypothesis that we confirmed by molecular dynamics simulations. Each monomer of the Aqy1 tetramers forms a channel whose walls consist mostly of hydrophilic residues, transporting through the selectivity filter containing Arg-227, His-212, Phe-92, and Ala-221, and the two conserved Asn-Pro-Ala (NPA) motifs containing asparagines 224 and 112. In summary, not all fungal aquaporins share the same gating mechanism by a tyrosine residue in the N-terminus, and the structural analysis in the present study should aid our understanding of aquaporin structure and its functional implications.     

Activities of Urease and Nickel Uptake of Helicobacter pylori Proteins are Media- and Host-Dependent

Background:  Nickel-dependent urease activity and nickel supply are essential for successful colonization of Helicobacter pylori in the acidic environment of the human stomach. A comparison of media effects on these two activities have never been carried out. Additionally to H. pylori we cultivated an Escherichia coli strain expressing the urease and the nickel transporter NixA of H. pylori on the same four media and measured in all cases urease and nickel uptake activity.
Aim:  To compare nickel uptake and urease activity on an inter- and intraspecies level.
Results:  In H. pylori nickel uptake (four to 200 times) and urease activities (400 to 30,000 times) were found to be much higher in comparison to the tested E. coli strain after growth on all media. These differences could not be explained by reduced protein amounts in the heterologous host E. coli . On which media the two bacteria extracted most of the nickel were organism-dependent: E. coli on Brucella Broth, H. pylori on Trypticase Soy Broth, and Minimal Media.
Conclusion:  H. pylori took nickel much more efficiently up than E. coli . The observed differences in urease activity are most likely due to additional protein components absent in the recombinant E. coli strain. The observed variety in nickel uptake and urease activities on the different media in the same organism depended on the intrinsic nickel content and chelating capacities of media components. Different culture conditions may lead to varying results; generalizations should be concluded only after excluding their media dependence.     

Mapping of the amino acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin

By using a photoactivatable analog of 11-cis-retinal in rhodopsin, we have previously identified the amino acids Phe-115, Ala-117, Glu-122, Trp-126, Ser-127, and Trp-265 as major sites of cross-linking to the chromophore. To further investigate the amino acids that interact with retinal, we have now used site-directed mutagenesis to replace a variety of amino acids in the membrane-embedded helices in bovine rhodopsin, including those that were indicated by cross-linking studies. The mutant rhodopsin genes were expressed in monkey kidney cells (COS-1) and purified. The mutant proteins were studied for their spectroscopic properties and their ability to activate transducin. Substitution of the two amino acids, Trp-265 and Glu-122 by Tyr, Phe, and Ala and by Gln, Asp and Ala, respectively, resulted in blue-shifted (20-30 nm) chromophore, and substitution of Trp-265 by Ala resulted in marked reduction in the extent of chromophore regeneration. Light-dependent bleaching behavior was significantly altered in Ala-117----Phe, Trp-265----Phe, Ala, and Ala-292----Asp mutants. Transducin activation was reduced in these mutants, in particular Trp-265 mutants, as well as in Glu-122----Gln, Trp-126----Leu (Ala), Pro-267----Ala (Asn, Ser), and Tyr-268----Phe mutants. These findings indicate that Trp-265 is located close to retinal and Glu-122, Trp-126, and probably Tyr-268 are also likely to be near retinal.     

Identification of simian virus 40 T-antigen residues important for specific and nonspecific binding to DNA and for helicase activity.

We have previously identified three regions (called elements) in the DNA-binding domain of simian virus 40 large tumor (T) antigen which are critical for binding of the protein to the recognition pentanucleotides GAGGC at the viral replication origin. These are elements A (residues 147 to 159), B1 (185 to 187), and B2 (203 to 207). In this study, we generated mutants of simian virus 40 in order to make single-point substitution mutations at nearly every site in these three elements. Each mutation was tested for its effect on virus replication, and T antigen was produced from all replication-negative mutants. The mutant proteins were assayed for binding to several different DNA substrates and for helicase activity. We found that within each element, mutations at some sites had major effects on DNA binding while mutations at other sites had moderate, mild, or minimal effects, suggesting that some residues are more important than others in mediating DNA binding. Furthermore, we provide evidence that certain residues in elements A and B2 (Ala-149, Phe-159, and His-203) participate in nonspecific double-stranded and helicase substrate (single-stranded) DNA binding while others (Ser-147, Ser-152, Asn-153, Thr-155, Arg-204, Val-205, and Ala-207) are involved in sequence-specific binding at the origin. The residues in element B1 (primarily Ser-185 and His-187) take part only in nonspecific DNA binding. The amino acids important for nonspecific DNA binding are also required for helicase activity, and we hypothesize that they make contact with the sugar-phosphate backbone of DNA. On the other hand, those involved in sequence-specific binding are not needed for helicase activity. Finally, our analysis showed that three residues (Asn-153 and Thr-155 in element A and Arg-204 in element B2) may be the most important for sequence-specific binding. They are likely to make direct or indirect contacts with the pentanucleotide sequences at the origin.     

Characterization of the amino acids involved in substrate specificity of methionine sulfoxide reductase A

Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide (MetSO) back to methionine. In vivo, Msrs are essential in protecting cells against oxidative damages on proteins and in the virulence of some bacteria. There exists two structurally unrelated classes of Msrs. MsrAs are stereo-specific toward the S epimer on the sulfur of the sulfoxide, whereas MsrBs are specific toward the R isomer. Both classes of Msrs display a similar catalytic mechanism of sulfoxide reduction by thiols via the sulfenic acid chemistry and a better affinity for protein-bound MetSO than for free MetSO. Recently, the role of the amino acids implicated in the catalysis of the reductase step of Neisseria meningitidis MsrA was determined. In the present study, the invariant amino acids potentially involved in substrate binding, i.e. Phe-52, Trp-53, Asp-129, His-186, Tyr-189, and Tyr-197, were substituted. The catalytic parameters under steady-state conditions and of the reductase step of the mutated MsrAs were determined and compared with those of the wild type. Altogether, the results support the presence of at least two binding subsites. The first one, whose contribution is major in the efficiency of the reductase step and in which the epsilon-methyl group of MetSO binds, is the hydrophobic pocket formed by Phe-52 and Trp-53, the position of the indole ring being stabilized by interactions with His-186 and Tyr-189. The second subsite composed of Asp-129 and Tyr-197 contributes to the binding of the main chain of the substrate but to a lesser extent.     

Biochemical Characterization and Structural Analysis of a Bifunctional Cellulase/Xylanase from Clostridium thermocellum

We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.     

Mutations in the small subunit of cyanobacterial ribulose-bisphosphate carboxylase/oxygenase that modulate interactions with large subunits

In the cyanobacterium Anacystis nidulans (Synechococcus PCC6301), ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) is composed of eight large subunits and eight small subunits. There are three regions of the small subunit that contain amino acids that are conserved throughout evolution, from bacteria to higher plants. Since the function of the small subunit is not fully understood, site-directed mutagenesis was performed on highly conserved residues in the first and second conserved regions. Ser-16, Pro-19, Leu-21, and Tyr-54 were replaced by Asp-16, His-19, Glu-21, and Ser-54, respectively. Crude extracts containing the recombinant His-19 mutant enzyme indicated that there was little effect on either Rbu-P2 carboxylase activity or interactions between large and small subunits. However, the Asp-16, Glu-21, and Ser-54 mutations showed effects on Rbu-P2 carboxylase activity and the interaction between large and small subunits. The large and small subunits of the Asp-16, Glu-21, and Ser-54 enzymes were found to dissociate during nondenaturing gel electrophoresis or sucrose density gradient centrifugation. However, the dissociated small subunits remained functional and were capable of reconstituting Rbu-P2 carboxylase activity when added to large subunits. These results indicated that Ser-16, Leu-21, and Tyr-54 might play an important role in interactions between large and small subunits of the A. nidulans enzyme.     

Contribution of dppA to urease activity in Helicobacter pylori 26695

BACKGROUND: The gastric pathogen Helicobacter pylori produces urease in amounts up to 10% of its cell protein. This enzyme, which catalyzes the hydrolysis of urea to ammonia and carbon dioxide, protects the bacterium from gastric acid. Urease, a nickel metalloenzyme, requires active uptake of nickel ions from the environment to maintain its activity. NixA is a nickel transport protein that resides in the cytoplasmic membrane. Mutation of nixA significantly reduces but does not abolish urease activity, strongly suggesting the presence of a second transporter. We postulated that the dipeptide permease (dpp) genes that are homologous to the nik operon of Escherichia coli could be a second nickel transporter. The predicted Dpp polypeptides DppA, DppC, and DppD of H. pylori share approximately 40%, 53%, and 56% amino acid sequence identity with their respective E. coli homologs. METHODS: A mutation in dppA, constructed by insertional inactivation with a chloramphenicol resistance cassette, was introduced by allelic exchange into H. pylori strain 26695. RESULTS: When compared to the parental strain, urease activity was not decreased in a dppA mutant. CONCLUSIONS: DppA does not contribute to the synthesis of catalytically active urease in H. pylori 26695 and is likely not a nickel importer in H. pylori.     

Mutagenesis of the Sauromatum guttatum alternative oxidase reveals features important for oxygen binding and catalysis

The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in mitochondria of all higher plants studied to date. To investigate the role of highly conserved amino acid residues in catalysis we have expressed site-directed mutants of Cys-172, Thr-179, Trp-206, Tyr-253, and Tyr-299 in AOX in the yeast Schizosaccharomyces pombe. Assessment of AOX activity in isolated yeast mitochondria reveals that mutagenesis of Trp-206 to phenylalanine or tyrosine abolishes activity, in contrast to that observed with either Tyr-253 or 299 both mutants of which retained activity. None of the mutants exhibited sensitivity to Q-like inhibitors that differed significantly from the wild type AOX. Interestingly, however, mutagenesis of Thr-179 or Cys-172 (a residue implicated in AOX regulation by α-keto acids) to alanine not only resulted in a decrease of maximum AOX activity but also caused a significant increase in the enzyme's affinity for oxygen (4- and 2-fold, respectively). These results provide important new insights in the mechanism of AOX catalysis and regulation by pyruvate.     

Location and functional significance of retinol-binding sites on the serine/threonine kinase, c-Raf

Redox activations of serine/threonine kinases represent alternate pathways in which vitamin A plays a crucial co-factor role. Vitamin A binds the zinc finger domain of c-Raf with nanomolar affinity. The retinoid-binding site has been mapped within this structure by scanning mutagenesis. The deduced contact sites were found anchored on Phe-8, counting from the 1st conserved histidine of the zinc finger. These sites agreed with contact amino acids identified by computational docking. The boundaries of a related binding pocket were identified by mutagenesis and partially confirmed by docking trials in the protein kinase C-alpha C1A zinc finger. They comprised Phe-7, Phe-8, and Trp-22. This trio was absent from the alphaC1B domain, explaining why the latter did not bind retinol. Reconfiguring at a minimum the two corresponding amino acids of alphaC1B, Thr-7 and Tyr-22, to conform to alphaC1A converted this domain to a binder. Deletion of the predicted retinoid-binding site in the full-length molecule created a mutant c-Raf that was deficient in retinol-dependent redox activation but fully responsive to epidermal growth factor. Our findings indicate that ligation of retinol to a specific site embedded in the regulatory domain is an important feature of c-Raf regulation in the redox pathway.     

The proton channel of the energy-transducing nicotinamide nucleotide transhydrogenase of Escherichia coli

The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple direct hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III to proton translocation by the membrane-intercalated domain II. To delineate the proton channel of the enzyme, 25 conserved and semiconserved prototropic amino acid residues of domain II of the Escherichia coli transhydrogenase were mutated, and the mutant enzymes were assayed for transhydrogenation from NADPH to an NAD analogue and for the coupled outward proton translocation. The results confirmed the previous findings of others and ourselves on the essential roles of three amino acid residues and identified another essential residue. Three of these amino acids, His-91, Ser-139, and Asn-222, occur in three separate membrane-spanning alpha helices of domain II of the beta subunit of the enzyme. Another residue, Asp-213, is probably located in a cytosolic-side loop that connects to the alpha helix bearing Asn-222. It is proposed that the three helices bearing His-91, Ser-139, and Asn-222 come together, possibly with another highly conserved alpha helix to form a four-helix bundle proton channel and that Asp-213 serves to conduct protons between the channel and domain III where NADPH binding energy is used via protein conformation change to initiate outward proton translocation.     

Insights into how CUB domains can exert specific functions while sharing a common fold: conserved and specific features of the CUB1 domain contribute to the molecular basis of procollagen C-proteinase enhancer-1 activity

Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies.     

Analysis of the membrane topology of the acid-sensing ion channel 2a

Acid-sensing ion channels, or ASICs, are members of the amiloride-sensitive cationic channel superfamily that are predicted to have intracellular amino and carboxyl termini and two transmembrane domains connected by a large extracellular loop. This prediction comes from biochemical studies of the mammalian epithelial sodium channels where glycosylation mutants identified the extracellular regions of the channel and a combination of antibody sensitivity and protease action substantiated the intracellular nature of the amino and carboxyl termini. However, although there are highly conserved regions within the different cation channel family members, membrane topology prediction programs provide several alternative structures for the ASICs. Thus, we used glycosylation studies to define the actual membrane topology of the ASIC2a subtype. We deleted the five predicted endogenous asparagine-linked glycosylation sites (Asn-Xaa-(Ser/Thr)) at Asn-22, Asn-365, Asn-392, Asn-478, and Asn-487 to map the extracellular topology. We then introduced exogenous asparagine-linked glycosylation sites at Lys-4, Pro-37, Arg-63, Tyr-67, His-72, Ala-81, Tyr-414, Tyr-423, and Tyr-453 to define the transmembrane domain borders. Finally, we used cell permeabilization studies to confirm the intracellular amino termini of ASIC2a. The data show that Asn-365 and Asn-392 are extracellular and that the introduction of asparagine-linked glycosylation sites at His-72, Ala-81, Tyr-414, and Tyr-423 leads to an increase in molecular mass consistent with an extracellular apposition. In addition, heterologous expression of ASIC2a requires membrane permeabilization for antibody staining. These data confirm the membrane topology prediction that the ASIC2a subtype consists of intracellular amino and carboxyl termini and two transmembrane domains connected by a large extracellular loop.     

Interaction between collagen and the alpha(2) I-domain of integrin alpha(2)beta(1). Critical role of conserved residues in the metal ion-dependent adhesion site (MIDAS) region.

A docking model of the alpha(2) I-domain and collagen has been proposed based on their crystal structures (Emsley, J., King, S., Bergelson, J., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). In this model, several amino acid residues in the I-domain make direct contact with collagen (Asn-154, Asp-219, Leu-220, Glu-256, His-258, Tyr-285, Asn-289, Leu-291, Asn-295, and Lys-298), and the protruding C-helix of alpha(2) (residues 284-288) determines ligand specificity. Because most of the proposed critical residues are not conserved, different I-domains are predicted to bind to collagen differently. We found that deleting the entire C-helix or mutating the predicted critical residues had no effect on collagen binding to whole alpha(2)beta(1), with the exception that mutating Asn-154, Asp-219, and His-258 had a moderate effect. We performed further studies and found that mutating the conserved surface-exposed residues in the metal ion-dependent adhesion site (MIDAS) (Tyr-157 and Gln-215) significantly blocks collagen binding. We have revised the docking model based on the mutagenesis data. In the revised model, conserved Tyr-157 makes contact with collagen in addition to the previously proposed Asn-154, Asp-219, His-258, and Tyr-285 residues. These results suggest that the collagen-binding I-domains (e.g. alpha(1), alpha(2), and alpha(10)) bind to collagen in a similar fashion.     

Oxygen-dependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese

During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.