Autocatalytic maturation of the Tat-dependent halophilic subtilase Nep produced by the archaeon Natrialba magadii

Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and are salt dependent for structural integrity and functionality. In contrast to bacterial subtilases, the maturation mechanism of halolysins has not been addressed. The halolysin Nep is secreted by the alkaliphilic haloarchaeon Natrialba magadii, and the recombinant active enzyme has been synthesized in Haloferax volcanii. Nep contains an N-terminal signal peptide with the typical Tat consensus motif (GRRSVL), an N-terminal propeptide, the protease domain, and a C-terminal domain. In this study, we used Nep as a model protease to examine the secretion and maturation of halolysins by using genetic and biochemical approaches. Mutant variants of Nep were constructed by site-directed mutagenesis and expressed in H. volcanii, which were then analyzed by protease activity and Western blotting. The Tat dependence of Nep secretion was demonstrated in Nep RR/KK variants containing double lysine (KK) in place of the twin arginines (RR), in which Nep remained cell associated and the extracellular activity was undetectable. High-molecular-mass Nep polypeptides without protease activity were detected as cell associated and extracellularly in the Nep S/A variant, in which the catalytic serine 352 had been changed by alanine, indicating that Nep protease activity was needed for precursor processing and activation. Nep NSN 1-2 containing a modification in two potential cleavage sites for signal peptidase I (ASA) was not efficiently processed and activated. This study examined for the first time the secretion and maturation of a Tat-dependent halophilic subtilase.     

Extracellular protease of Natrialba magadii: purification and biochemical characterization

A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60°C in the presence of 1.5 M NaCl. The enzyme was stable and had a broad pH profile (6–12) with an optimum pH of 8–10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated. Received: October 1, 1999 / Accepted: February 1, 2000     

Two immunologically distinct types of protofilaments can be identified in Natrialba magadii flagella

A rabbit antiserum to the 15-kDa acetylcholinesterase toxin neutralised the lethal effect of the 15-kDa toxin of Aeromonas hydrophila when injected into trout. However, immunisation of fish with the 15-kDa toxoid failed to induce an antibody response, and a higher molecular mass form of this toxin was purified from the extracellular products with the aim of inducing an immune response in fish. The optimal conditions for production of extracellular products by A. hydrophila strain B32 were studied to increase the concentration of this protoxin. The extracellular products were fractionated by molecular exclusion chromatography to yield a purified protoxin with an estimated molecular mass of 45 kDa by SDS-PAGE and which gave a positive reaction in Western blotting with the rabbit anti-15-kDa toxin serum. Since the 45-kDa protoxin showed lower specific acetylcholinesterase activity than the active 15-kDa toxin, the behaviour of the active site was studied using specific inhibitors. This 45-kDa protoxin was 13.3-fold less toxic than the 15-kDa toxin and induced antibody production in fish.     

Effect of nutritional conditions on extracellular protease production by the haloalkaliphilic archaeon Natrialba magadii

AIMS: The effect of various nitrogen sources and nutritional starvation was examined on the production of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii. METHODS AND RESULTS: Cell growth and proteolytic activity were measured in cells grown with different nitrogen sources. Proteolytic activity was produced in complex and easily metabolized nitrogen sources such as yeast extract, casein and casamino acids; meanwhile, ammonium repressed enzyme production. The time course and amount of protease accumulated showed an inverse correlation with growth rate and nutrient concentration. Starvation did not induce extracellular protease production. CONCLUSION: The accumulation of Nab. magadii extracellular protease is stimulated by nutrient limitation and slow growth rate indicating that it is probably induced in response to a deficit in the energetic status of the cells. Nutritional starvation did not induce protease accumulation suggesting that de novo synthesis of this protease and/or factor/s necessary for its activation are required. This enzyme may be regulated by nitrogen catabolite repression and it does not require protein substrates for induction. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the basic knowledge on protease regulation in haloalkaliphilic archaea and will help to optimize the production of this extremozyme for biotechnological applications such as protease-catalysed peptide synthesis.     

Natrialba magadii virus phiCh1: first complete nucleotide sequence and functional organization of a virus infecting a haloalkaliphilic archaeon

The double-stranded (ds)DNA virus phiCh1 infects the haloalkaliphilic archaeon Natrialba magadii. The complete DNA sequence of 58 498 bp of the temperate virus was established, and the probable functions of 21 of 98 phiCh1-encoded open reading frames (ORFs) have been assigned. This knowledge has been used to propose functional modules each required for specific functions during virus development. The phiCh1 DNA is terminally redundant and circularly permuted and therefore appears to be packaged by the so-called headful mechanism. The presence of ORFs encoding homologues of proteins involved in plasmid replication as well as experimental evidence indicate a plasmid-mediated replication strategy of the virus. Results from nanosequencing of virion components suggest covalent cross-linking of monomers of at least one of the structural proteins during virus maturation. A comparison of the phiCh1 genome with the partly sequenced genome of Halobacterium salinarum virus phiH revealed a close relationship between the two viruses, although their host organisms live in distinct environments with respect to the different pH values required for growth.     

Chemical shifts in denatured proteins: Resonance assignments for denatured ubiquitin and comparisons with other denatured proteins

Chemical shift assignment is reported for the protein ubiquitin denatured in 8M urea at pH 2. The variations in ¹⁵N chemical shifts of three different proteins (ubiquitin, disulfide reduced, carboxymethylated lysozyme, all-Ala--lactalbumin), all without disulfides and denatured in 8M urea at pH 2 are compared to `random coil shifts' of small model peptides (Braun et al., 1994) and to the averaged native chemical shifts taken from the BMRB database. Both parameterizations show a remarkable agreement with the averaged measured ¹⁵N chemical shifts in the three denatured proteins. Detailed analysis of these experimental ¹⁵N chemical shifts provides an estimate of the influence of nearest neighbors and conformational preferences on the chemical shift and provides a direct means to identify non-random structural preferences in denatured proteins.     

Nucleoside diphosphate kinase from haloalkaliphilic archaeon Natronobacterium magadii: purification and characterization

An ATP-binding protein from the haloalkaliphilic archaeon Natronobacterium magadii was purified and characterized by affinity chromatography on ATP-agarose and by fast protein liquid chromatography (FPLC) on a Mono Q column. The N-terminal 20 amino acid sequence of the kinase showed a strong sequence similarity of this protein with nucleoside diphosphate (NDP) kinases from different organisms and, accordingly, we believe that this protein is a nucleoside diphosphate kinase, an enzyme whose main function is to exchange γ-phosphates between nucleoside triphosphates and diphosphates. Comparison of the molecular weights of the NDP kinase monomer determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (23 000) and of the oligomer determined by sedimentation equilibrium experiments (125 000) indicated that the oligomer is a hexamer. The enzyme was autophosphorylated in the presence of [γ-³²P]ATP, and Mg²⁺ was required for the incorporation of phosphate. The kinase preserved the ability to transfer γ-phosphate from ATP to GDP in the range of NaCl concentration from 90 mM to 3.5 M and in the range of pH from 5 to 12. It was found and confirmed by Western blotting that this kinase is one of the proteins that bind specifically to natronobacterial flagellins. NDP kinase from haloalkaliphiles appeared to be simple to purify and to be a suitable enzyme for studies of structure and stability compared with NDP kinases from mesophilic organisms. Received: December 3, 1997 / Accepted: January 29, 1998     

Structural stability of halophilic proteins

An examination of halobacterial amino acids exchanges as they appear in the known Spirulina platensis [2Fe-2S] ferredoxin tertiary structure indicated that most of the additional acidic residues of the halophiles occurred on the external surface of the alga structure; however, further negative changes were not placed in the ferredoxin active site region. A statistical investigation of the amino acid compositions of seven halophile and nonhalophile protein counterparts indicated that the bulkiness of amino acids used by halophiles is considerably reduced and that the overall hydrophobicity of halophilic and non halophilic molecules was essentially the same. It is suggested that the principal mode of structural stabilization for halophilic proteins is effective competition with the cytoplasmic salt for water through utilization of many external carboxyl groups of glutamic and aspartic acids. A reduction is residue bulkiness would prevent inactivation in the presence of the high molarity, antichaotropic KCl. Halophilic functionality is preserved through avoidance of additional negative charge at the active site surface.     

Overexpression of wild-type and mutant NDP kinase in Dictyostelium discoideum

Nucleoside diphosphate (NDP) kinase has a central role in the synthesis of (deoxy-)trinucleotides. In addition, mutations in the gene encoding NDP kinase have been shown to have important consequences for Drosophila development and mammalian tumorogenesis. We have overexpressed, in Dictyostelium discoideum, a genomic clone encoding the enzyme NDP kinase. The concomitant increase in the levels of RNA and enzyme activity identifies a 5′ non-coding genomic region of 0.9 kb as being the complete promoter region. Overexpression of wild-type NDP kinase has no effect on development. This is also true for an inactive mutant H122C that does not have a dominant inhibitor effect. Overexpression of the P105G mutant NDP kinase, which is known to be affected in its stability in vitro, only leads to a small increase in total NDP-kinase activity. Thermal and chemical denaturation experiments demonstrate the formation of hexameric hybrids between wild-type and mutant monomers.     

Active site comparisons highlight structural similarities between myosin and other P-loop proteins.

The phosphate binding loop (P-loop) is a common feature of a large number of enzymes that bind nucleotide whose consensus sequence is often used as a fingerprint for identifying new members of this group. We review here the binding sites of nine purine nucleotide binding proteins, with a focus on their relationship to the active site of myosin. This demonstrates that there is considerable conversation in the distribution and nature of the ligands that coordinate the triphosphate moiety. This comparison further suggests that at least myosin and the G-proteins utilize a similar mechanism for nucleotide hydrolysis.     

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.     

Structure comparisons of Aedes albopictus densovirus with other parvoviruses

Parvoviridae is a family of the smallest viruses known with a wide variety of hosts. The capsid structure of the Aedes albopictus C6/36 cell densovirus (C6/36 DNV) at 1.2-nm resolution was obtained by electron cryomicroscopy (cryoEM) and three-dimensional (3D) image reconstruction. Structure comparisons between the C6/36 DNV and other parvoviruses reveal that the degree of structural similarity between C6/36 DNV and the human parvovirus B19 is higher than that between C6/36 DNV and other insect parvoviruses. The amino acid sequence comparisons of structural and non-structural proteins also reveal higher levels of similarity between C6/36 DNV and parvovirus B19 than those between C6/36 DNV and other parvoviruses. These findings indicate that C6/36 DNV is closely related to the human virus B19, and the former might evolve from the human species other than from other insect viruses.     

Structure comparisons of Aedes albopictus densovirus with other parvoviruses

Parvoviridae is a family of the smallest viruses known with a wide variety of hosts. The capsid structure of the Aedes albopictus C6/36 cell densovirus (C6/36 DNV) at 1.2-nm resolution was obtained by elec-tron cryomicroscopy (cryoEM) and three-dimensional (3D) image reconstruction. Structure compari-sons between the C6/36 DNV and other parvoviruses reveal that the degree of structural similarity be-tween C6/36 DNV and the human parvovirus B19 is higher than that between C6/36 DNV and other in-sect parvoviruses. The amino acid sequence comparisons of structural and non-structural proteins also reveal higher levels of similarity between C6/36 DNV and parvovirus B19 than those between C6/36 DNV and other parvoviruses. These findings indicate that C6/36 DNV is closely related to the human virus B19, and the former might evolve from the human species other than from other insect viruses.     

Nm23-H1/NDP kinase folding intermediates and cancer: a hypothesis

The Nm23-H1/nucleoside diphosphate (NDP) kinase A is a metastasis suppressor, besides its enzymatic activity. The mutant S120G has been found in high-grade neuroblastomas. The mutant protein, once denatured in urea, is unable to refold in vitro. A size-exclusion chromatography analysis of the folding/association pathway showed that recombinant wild-type and S120G mutant human Nm23-H1/NDP kinase A unfold and refold passing through a molten globule state while typical hexameric NDP kinases unfold without dissociated species and refold through a native monomeric intermediate. A survey of the recent literature showed that several proteins involved in cancer, and their mutants, are marginally stable, like the wild-type Nm23-H1/NDP kinase A, or are misfolded, like its S120G mutant. We therefore suggest that the low thermodynamic stability and the folding intermediate of the Nm23-H1/NDP kinase A may be necessary for its regulatory properties.