Microbial transformations of steroids-XI. Progesterone transformation by Streptomyces roseochromogenes-purification and characterisation of the 16alpha-hydroxylase system

Streptomyces roseochromogenes, NCIB 10984, contains a cytochrome P450 which, in conjunction with two indigenous electron transfer proteins, roseoredoxin and roseoredoxin reductase, hydroxylates exogenous progesterone firstly to 16alpha-hydroxyprogesterone and thereafter in a second phase bioconversion to 2beta,16alpha-dihydroxyprogesterone. The progesterone 16alpha-hydroxylase P450 and the two electron transfer proteins have been purified to homogeneity. A reconstituted incubation containing these three purified proteins and NADH, the natural electron donor, produced identical hydroxy-progesterone metabolites as in intact cells. Peroxy and hydroperoxy compounds act in a shortened form of the cycle known as the 'peroxide shunt' by replacing the natural pathway requirement for the electron donor NADH, the electron transfer proteins and molecular O2, the terminal electron acceptor. In an NaIO4 supported incubation, the initial rate of progesterone hydroxylation was marginally higher (1.62 mmol progesterone/mmol P-450/h) than in the reconstituted natural incubation (1.18 mmol progesterone/mmol P-450/h) but the product yield was significantly lower, 0.45 mol hydroxyprogesterone produced/mol P-450 compared to 6.0 mol hydroxyprogesterone produced/mol P-450. These yield data show that in the reconstituted natural pathway, progesterone 16alpha-hydroxylase P450 supports multiple rounds of hydroxylation in contrast to a likely single oxygenation by a minority of P450s in the peroxide shunt pathway.     

Regulation by S-nitrosylation of protein post-translational modification

Protein post-translational modification by S-nitrosylation conveys a ubiquitous influence of nitric oxide on signal transduction in eukaryotic cells. The wide functional purview of S-nitrosylation reflects in part the regulation by S-nitrosylation of the principal protein post-translational modifications that play a role in cell signaling, including phosphorylation, acetylation, ubiquitylation and related modifications, palmitoylation, and alternative Cys-based redox modifications. In this minireview, we discuss the mechanisms through which S-nitrosylation exerts its broad pleiotropic influence on protein post-translational modification.     

Regulation of CRAC channels by protein interactions and post-translational modification

Store-operated Ca²⁺ entry (SOCE) is a widespread mechanism to elevate the intracellular Ca²⁺ concentrations and stimulate downstream signaling pathways affecting proliferation, secretion, differentiation and death in different cell types. In immune cells, immune receptor stimulation induces intracellular Ca²⁺ store depletion that subsequently activates Ca²⁺-release-activated-Ca²⁺ (CRAC) channels, a prototype of store-operated Ca²⁺ (SOC) channels. CRAC channel opening leads to activation of diverse downstream signaling pathways affecting proliferation, differentiation, cytokine production and cell death. Recent identification of STIM1 as the endoplasmic reticulum Ca²⁺ sensor and Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tools to dissect the mechanism of activation and regulation of CRAC channels. In this review, we discuss the recent advances in understanding the associating partners and posttranslational modifications of Orai1 and STIM1 proteins that regulate diverse aspects of CRAC channel function.     

Barley lipid transfer protein, LTP1, contains a new type of lipid-like post-translational modification.

In plants a group of proteins termed nonspecific lipid transfer proteins are found. These proteins bind and catalyze transfer of lipids in vitro, but their in vivo function is unknown. They have been suggested to be involved in different aspects of plant physiology and cell biology, including the formation of cutin and involvement in stress and pathogen responses, but there is yet no direct demonstration of an in vivo function. We have found and characterized a novel post-translational modification of the barley nonspecific lipid transfer protein, LTP1. The protein-modification bond is of a new type in which an aspartic acid in LTP1 is bound to the modification through what most likely is an ester bond. The chemical structure of the modification has been characterized by means of two-dimensional homo- and heteronuclear nuclear magnetic resonance spectroscopy as well as mass spectrometry and is found to be lipid-like in nature. The modification does not resemble any standard lipid post-translational modification but is similar to a compound with known antimicrobial activity.     

Factors influencing expression and post-translational modification of recombinant protein C.

Factors affecting the production of recombinant human protein C were investigated. When recombinant cells producing human protein C were cultured with microcarrier in two different scales, we found that (1) as cells grew to confluence, specific productivity of the protein was decreased and that (2) the efficiency of gamma-carboxylation of the generated protein C was lower in a larger culture than in a smaller culture. Higher cell density was shown to influence the specific productivity unfavorably. On the other hand, the amount of oxygen supply as demonstrated by oxygen consumption rate in the Opticell culture system correlated well with the efficiency of gamma-carboxylation, suggesting that oxygen metabolism is somehow implicated in the post-translational modification of recombinant protein C.     

Dramatic modulation of electron transfer in protein complexes by crosslinking.

The transfer of electrons between proteins is an essential step in biological energy production. Two protein redox partners are often artificially crosslinked to investigate the poorly understood mechanism by which they interact. To better understand the effect of crosslinking on electron transfer rates, we have constructed dimers of azurin by crosslinking the monomers. The measured electron exchange rates, combined with crystal structures of the dimers, demonstrate that the length of the linker can have a dramatic effect on the structure of the dimer and the electron transfer rate. The presence of ordered water molecules in the protein-protein interface may considerably influence the electronic coupling between redox centers.     

Aromatic hydroxylation and sulfation of 5-hydroxyflavone by Streptomyces fulvissimus.

The conversion of 5-hydroxyflavone by various microorganisms was studied. Among them, Streptomyces fulvissimus was the sole microbe which produced a new polar metabolite from 5-hydroxyflavone in addition to 5,4-dihydoxy- and 5,3,4-trihydroxyflavone. The structure of this polar metabolite was determined to be 5,4-dihydroxyflavone-4-sulfate on the basis of mass, infrared, and nuclear magnetic resonance spectroscopies. These results demonstrate that S. fulvissimus catalyzes sulfation at the 4 position of 4,5-dihydroxyflavone.     

Microbial transformations of steroids--I. Rare transformations of progesterone by Apiocrea chrysosperma

When Apiocrea chrysosperma is incubated with progesterone for 7 days in a peptone, yeast-extract medium, eight major metabolites are produced. Each compound has been purified and its structure determined by high-field 1D and 2D 1H nuclear magnetic resonance (NMR) spectroscopy. A clear synthetic pattern is recognisable. The products have been formed by multiple transformation reactions, usually double hydroxylations. Seven compounds are tertiary alcohols in which the hydroxyl group is located on the underside of the progesterone skeleton at either the axial 9 alpha- or the axial 14 alpha-site. One compound has hydroxyl groups at both these sites. Five metabolites are also secondary progesterone alcohols, the hydroxyl groups being at the 6 beta-, 15 alpha- or 15 beta-sites. Two compounds are monohydroxy metabolites; one is dehydrogenated in ring B and the other has lost the pregnane side-chain. The structures of the eight metabolites are 6 beta, 9 alpha-dihydroxyprogesterone; 6 beta, 14 alpha-dihydroxyprogesterone; 9 alpha, 14 alpha-dihydroxyprogesterone; 9 alpha, 15 beta-dihydroxyprogesterone, 14 alpha, 15 alpha-dihydroxyprogesterone; 14 alpha, 15 beta-dihydroxyprogesterone; 14 alpha-hydroxypregna-4,6-diene-3,20-dione and 15 alpha-hydroxyandrostene-3,17-dione. All compounds, except the last one, are biologically rare because they are not products of mammalian progesterone or androstenedione metabolism. They would be difficult to synthesise chemically. We believe that the compounds, 9 alpha, 15 beta-dihydroxyprogesterone; 14 alpha, 15 alpha-dihydroxyprogesterone and 14 alpha-hydroxypregn-4,6-diene-3,20-dione, have not been reported previously as microbial transformation products of progesterone.     

A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification

Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neuro-degeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multi-protein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation.     

A multimeric model for murine anti-apoptotic protein Bcl-2 and structural insights for its regulation by post-translational modification.

A monomeric model for murine antiapoptotic protein Bcl-2 was constructed by comparative modeling with the software suite MPACK (EXDIS/DIAMOD/FANTOM) using human Bcl-xL as a template. The monomeric model shows that murine Bcl-2 is an all -helical protein with a central (helix 5) hydrophobic helix surrounded by amphipathic helices and an unstructured loop of 30 residues connecting helices 1 and 2. It has been previously shown that phosphorylation of Ser 70 located in this loop region regulates the anti-apoptotic activity of Bcl-2. Based on our current model, we propose that this phosphorylation may result in a conformational change that aids multimer formation. We constructed a model for the Bcl-2 homodimer based on the experimentally determined 3D structure of the Bcl-xL: Bad peptide complex. The model shows that it will require approximately a half turn in helix 2 to expose hydrophobic residues important for the formation of a multimer. Helices 5 and 6 of the monomeric subunit Bcl-2 have been proposed to form an ion-channel by associating with helices 5 and 6 of another monomeric subunit in the higher-order complex. In the multimeric model of Bcl-2, helices 5 and 6 of each subunit were placed distantly apart. From our model, we conclude that a global conformational change may be required to bring helices 5 and 6 together during ion-channel formation.Figure Hydrophobic interactions in the dimerization groove are shown. Helix 2' of monomer B interacts through residues V90, H91, L94, A97, G98, F101 and Y105 with the hydrophobic surface formed by residues in helices 3, 4, and 5 of the monomer A. Shown here is a lateral view of monomer A depicted in a surface model with hydrophobic regions in red. The backbone of the helix is shown using a neon representation in yellow and the interacting side chains are in blue.     

Reversible post-translational modification of proteins by nitrated fatty acids in vivo

Nitric oxide ((*)NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon beta to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC(50) of approximately 3 microM for both nitroalkenes, an IC(50) equivalent to the potent thiol oxidant peroxynitrite (ONOO(-)) and an IC(50) 30-fold less than H(2)O(2), indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking.     

Microbial transformations of steroids--III. Transformation of progesterone by Sepedonium ampullosporum

The 16 alpha-steroid hydroxylating fungus Sepedonium ampullosporum (CMI strain 203 033) transformed progesterone into 16 alpha-hydroxyprogesterone and four other major metabolites which have not been reported previously for this organism, 6 beta-hydroxyprogesterone, 17 alpha-hydroxyprogesterone, 16 alpha-hydroxyandrostenedione and 16-oxotestosterone (16-ketotestosterone). Among the minor metabolites we have been able to identify 15 alpha-hydroxyprogesterone. This compound has not been reported for S. ampullosporum. The conditions used for transformation had comparatively little effect on the relative proportions of products formed, 16 alpha-hydroxyprogesterone always being the predominant metabolite, but had a major effect on the total yields of metabolites isolatable. These findings suggest that one or more constitutive enzyme systems were responsible for the transformations.     

A post-translational modification, unrelated to hydroxylation, in the collagenous domain of nonhelical pro-alpha 2(I) procollagen chains secreted by chemically transformed hamster fibroblasts.

Transformed Syrian hamster embryo (NQT-SHE) fibroblasts do not synthesize the pro-alpha 1 subunit of type I procollagen, but secrete two modified forms of the pro-alpha 2(I) subunit that migrate more slowly than the normal chain during gel electrophoresis (Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826). By electrophoretic analysis of cyanogen bromide and V8 protease-derived peptides from the collagenous domains of intra- and extracellular pro-alpha 2(I) chains, we find that the modification occurs almost exclusively in secreted molecules, is located in the region spanned by the cyanogen bromide peptide CB3,5, and persists when hydroxylation is inhibited. Thus, modification is due to a post-translational reaction other than hydroxylation. The modified chains appear to be secreted in the denatured state since: 1) helical structures formed at 4 degrees C under acidic conditions were unstable under neutral conditions at 37 degrees C; 2) conditions that destabilize the type I procollagen helix and thus inhibit its secretion, i.e. inhibition of proline hydroxylation or incorporation of the proline analog cis-hydroxyproline, did not affect secretion of the modified chains. The time courses for secretion of nonhelical modified chains from NQT-SHE and of hydroxylated helical procollagen I from control cells, as a proportion of total collagen synthesized, were similar. Although cis-hydroxyproline did not inhibit the secretion of the modified chains, it induced their rapid intracellular degradation.     

Comparative studies on isoaccepting arginyl tRNAs from transformed cells and their utilization in post-translational protein modification.

-Comparative studies on the arginine-incorporating activity by the post-translational protein modification system in transformed or infected cells revealed that: (1) Transfer of arginine from arginyl-tRNA was better with the extract obtained from polyoma-infected baby mouse kidney cells than extracts from uninfected controls but similar efficiency of transfer was observed between the extracts of SV40-3T3 and 3T3 cells. Extracts of rat embryo cells transformed by the chemical carcinogen dimethylbenzanthracene, but not by 2-methylcholanthrene, had a higher arginine-incorporating activity than its normal counterpart. (2) On RPC-5 columns, the chromatographic profiles of isoaccepting arginyl-tRNAs derived from transformed or infected cells and their normal counterparts were similar. (3) The transfer of arginine from two species of isoaccepting arginyl-tRNAs of polyoma-infected baby mouse kidney cells was better than that from arginyl-tRNA of uninfected cells.     

Primary and secondary metabolism,and post-translational protein modifications,as portrayed by proteomic analysis of Streptomyces coelicolor

The newly sequenced genome of Streptomyces coelicolor is estimated to encode 7825 theoretical proteins. We have mapped approximately 10% of the theoretical proteome experimentally using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Products from 770 different genes were identified, and the types of proteins represented are discussed in terms of their annotated functional classes. An average of 1.2 proteins per gene was observed, indicating extensive post-translational regulation. Examples of modification by N-acetylation, adenylylation and proteolytic processing were characterized using mass spectrometry. Proteins from both primary and certain secondary metabolic pathways are strongly represented on the map, and a number of these enzymes were identified at more than one two-dimensional gel location. Post-translational modification mechanisms may therefore play a significant role in the regulation of these pathways. Unexpectedly, one of the enzymes for synthesis of the actinorhodin polyketide antibiotic appears to be located outside the cytoplasmic compartment, within the cell wall matrix. Of 20 gene clusters encoding enzymes characteristic of secondary metabolism, eight are represented on the proteome map, including three that specify the production of novel metabolites. This information will be valuable in the characterization of the new metabolites.     

Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase

The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for posttranslational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight beta-turn that houses the lysine residue targetted for biotinylation, and the beta-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct posttranslational modification.     

Control of electron transfer by protein dynamics in photosynthetic reaction centers

Abstract

Trehalose and glycerol are low molecular mass sugars/polyols that have found widespread use in the protection of native protein states, in both short- and long-term storage of biological materials, and as a means of understanding protein dynamics. These myriad uses are often attributed to their ability to form an amorphous glassy matrix. In glycerol, the glass is formed only at cryogenic temperatures, while in trehalose, the glass is formed at room temperature, but only upon dehydration of the sample. While much work has been carried out to elucidate a mechanistic view of how each of these matrices interact with proteins to provide stability, rarely have the effects of these two independent systems been directly compared to each other. This review aims to compile decades of research on how different glassy matrices affect two types of photosynthetic proteins: (i) the Type II bacterial reaction center from Rhodobacter sphaeroides and (ii) the Type I Photosystem I reaction center from cyanobacteria. By comparing aggregate data on electron transfer, protein structure, and protein dynamics, it appears that the effects of these two distinct matrices are remarkably similar. Both seem to cause a “tightening” of the solvation shell when in a glassy state, resulting in severely restricted conformational mobility of the protein and associated water molecules. Thus, trehalose appears to be able to mimic, at room temperature, nearly all of the effects on protein dynamics observed in low temperature glycerol glasses.