The initiation mess?

This review concerns the mechanisms which control initiation of chromosome replication in enterobacteria with respect to cell growth. Initiation control is commonly separated into positive and negative regulatory mechanisms. Four main points are advanced concerning these different aspects of initiation control. (i) The average concentration of the initiator protein DnaA is proportional to the origin concentration, i.e. the origin per cell mass ratio and, thus, inversely proportional to the very often used term of the 'initiation mass'. (ii) The time of initiation of chromosome replication in the cell cycle is set by DnaA protein accumulating to a threshold level, which in concert with a number of other factors allows for a co-operative formation of the initiation complex. (iii) The time of initiation is not determined by the interaction with these other factors or by the transient interaction between newly replicated origins ( oriC  ) and the cell surface. (iv) The aberrant initiation phenotype observed in various mutants, including dnaA (ts) mutants, might be due to a defective pre-initiation DnaA– oriC interaction or it might be due to a defect in the protection of newly initiated origins from reinitiation. Many of these points are discussed and evaluated in view of recent developments concerning the regulation of chromosome replication in Escherichia coli     

Inter‐ and intraspecific variation in body‐ and genome size in calanoid copepods from temperate and arctic waters

The tendency of ectotherms to get larger in the cold (Bergmann clines) has potentially great implications for individual performance and food web dynamics. The mechanistic drivers of this trend are not well understood, however. One fundamental question is to which extent variation in body size is attributed to variation in cell size, which again is related to genome size. In this study, we analyzed body and genome size in four species of marine calanoid copepods, Calanus finmarchicus, C. glacialis, C. hyperboreus and Paraeuchaeta norvegica, with populations from both south Norwegian fjords and the High Arctic. The Calanus species showed typical interspecific Bergmann clines, and we assessed whether they also displayed similar intraspecific variations—and if correlation between genome size and body size differed between species. There were considerable inter‐ as well as intraspecific variations in body size and genome size, with the northernmost populations having the largest values of both variables within each species. Positive intraspecific relationships suggest a functional link between body and genome size, although its adaptiveness has not been settled. Impact of additional drivers like phylogeny or specific adaptations, however, was suggested by striking divergences in body size – genome size ratios among species. Thus, C. glacialis and C. hyperboreus, had fairly similar genome size despite very different body size, while P. norvegica, of similar body size as C. hyperboreus, had the largest genome sizes ever recorded from copepods. The inter‐ and intraspecific latitudinal body size clines suggest that climate change may have major impact on body size composition of keystone species in marine planktonic food webs.     

Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli

Summary Increased synthesis of DnaA protein, obtained with plasmids carrying the dnaA gene controlled by the heat inducible pL promoter, stimulated initiation of replication from oriC about threefold. The overinitiation was determined both as an increase in copy number of a minichromosome and as an increase in chromosomal gene dosage of oriC proximal DNA. The additional replication forks which were initiated on the chromosome did not lead to an overall increase in DNA content. DNA/DNA hybridization showed an amplification encompassing less than a few hundred kilobases on each side of oriC. Kinetic studies showed that the overinitiation occurred very rapidly after the induction, and that the initiation frequency then decreased to a near normal frequency per oriC. The results indicate that the DnaA protein is one important factor in regulation of initiation of DNA replication from oriC.     

Arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 2: Optimization of P1 and N-aryl

A systematic study of anilines led to the discovery of a metabolically robust fluoroindoline replacement for the alkoxy aniline toxicophore in 1. Investigations of the P1 pocket resulted in the discovery of a wide tolerance of functionality leading to the discovery of 11 as a potent and selective inhibitor of cathepsin S.     

Synthesis and SAR of arylaminoethyl amides as noncovalent inhibitors of cathepsin S: P3 cyclic ethers

The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.     

Streptomyces coelicolor A3(2) CYP102 Protein,a Novel Fatty Acid Hydroxylase Encoded as a Heme Domain without an N-Terminal Redox Partner

The gene from Streptomyces coelicolor A3(2) encoding CYP102B1, a recently discovered CYP102 subfamily which exists solely as a single P450 heme domain, has been cloned, expressed in Escherichia coli, purified, characterized, and compared to its fusion protein family members. Purified reconstitution metabolism experiments with spinach ferredoxin, ferredoxin reductase, and NADPH revealed differences in the regio- and stereoselective metabolism of arachidonic acid compared to that of CYP102A1, exclusively producing 11,12-epoxyeicosa-5,8,14-trienoic acid in addition to the shared metabolites 18-hydroxy arachidonic acid and 14,15-epoxyeicosa-5,8,11-trienoic acid. Consequently, in order to elucidate the physiological function of CYP102B1, transposon mutagenesis was used to generate an S. coelicolor A3(2) strain lacking CYP102B1 activity and the phenotype was assessed.Streptomycetes produce a vast array of antibiotics applied in human and veterinary medicine and agriculture, as well as antiparasitic agents, herbicides, and pharmacologically active metabolites (e.g., immunosuppressants). Streptomycetes also catalyze numerous transformations of xenobiotics of industrial and environmental importance (1). The most significant of these biocatalytic reactions include aromatic and aliphatic hydroxylations, O and N dealkylations, N oxidation, and C-C coupling and fission catalyzed by heme-containing cytochrome P450 (CYP) enzyme systems (25). Streptomyces coelicolor A3(2) is the most-studied member of the genus in molecular genetic and biochemical investigations (1). Eighteen cytochrome P450, six ferredoxin, and four ferredoxin reductase genes were shown to be distributed across the linear chromosome, with nine P450 genes arranged in polycistronic organization with other genes (1, 14).Arguably, the most biochemically and structurally characterized CYP to date is CYP102A1 (P450 BM3) from Bacillus megaterium. CYP102A1 was first isolated as a fatty acid monooxygenase in the laboratory of Armand Fulco more than 20 years ago (16). It was established as a catalytically self-sufficient monooxygenase consisting of a CYP heme domain fused at the carboxy terminus to a flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD)-containing flavin reductase domain in a single polypeptide chain. It has been proposed that such architecture affords the CYP domain optimal catalytic activity in the turnover of substrates. Indeed, of all the CYPs characterized functionally, CYP102A1 exhibits the highest P450 turnover frequency, with rates of >15,000 min⁻¹ obtained in exogenous arachidonate hydroxylation (17, 18). Furthermore, CYP102A1-like P450 reductase fusion proteins have also been found in certain fungi, including Fusarium oxysporum (13) and Phanerochaete chrysosporium (3).CYP102B1 was the first member of a new CYP102 subfamily discovered outside Bacillus spp. (1) and is intriguing since it exists and functions as a single CYP protein domain. Here we report the cloning, expression, and characterization of CYP102B1 and demonstrate that the enzyme has activity in metabolizing arachidonic acid but with very different product profiles and with enzymatic rates orders of magnitude lower than those of CYP102A1. To address the question of the contribution of CYP102B1 to S. coelicolor A3(2) physiology, a CYP102B1 transposon mutant was generated and isolated and the phenotype of the subsequent mutant strains was analyzed.     

The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

Background

Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.

Results

Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.

Conclusions

Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP³⁵RIW motif of N-terminal Vpr.     

Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag

A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.