The N-terminal intrinsically disordered region of Ncb5or docks with the cytochrome b5 core to form a helical motif that is of ancient origin.

NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5 ) and cytochrome b5 reductase (b5 R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5 R domains (CS/b5 R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34 MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25-129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116 QMDWLKLTRT126 ) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes.

Effect of actinorhizal root exudates on the proteomes of Frankia soli NRRL B-16219, a strain colonizing the root tissues of its actinorhizal host via intercellular pathway.

Frankia and actinorhizal plants exchange signals in the rhizosphere leading to specific mutual recognition of partners and nitrogen-fixing nodule organogenesis. Frankia soli strain NRRL B-16219, from the Elaeagnus specificity group, colonizes the root tissues of its actinorhizal host through direct intercellular penetration of root epidermis cells and cortex. Here, we studied the early proteogenomic response of strain NRRL B-16219 to treatment with root exudates from compatible Elaeagnus angustifolia, and incompatible Ceanothus thyrsiflorus and Coriaria myrtifolia, host plants grown in nitrogen depleted hydroponic medium. Next-generation proteomics was used to identify the main Frankia proteins differentially expressed in response to the root exudates. No products of the nod genes present in B-16219 were detected. Proteins specifically upregulated in presence of E. angustifolia root exudates include those connected to nitrogen fixation and assimilation (glutamate synthetase, hydrogenase and squalene synthesis), respiration (oxidative phosphorylation and citric acid cycle pathways), oxidative stress (catalase, superoxide dismutase, and peroxidase), proteolysis (proteasome, protease, and peptidase) and plant cell wall degrading proteins involved in the depolymerization of celluloses (endoglucanase, glycosyltransferase, beta-mannanases, glycoside hydrolase and glycosyl hydrolase). Proteomic data obtained in this study will help link signaling molecules/factors to their biosynthetic pathways once those factors have been fully characterized.

Crystal structures of the naturally fused CS and cytochrome b5 reductase (b5R) domains of Ncb5or reveal an expanded CS fold, extensive CS-b5R interactions and productive binding of the NAD(P)+ nicotinamide ring.

Ncb5or (NADH-cytochrome b5 oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD-Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b5 (Cyb5A) and b5R (Cyb5R3) system, is of interest. A high-resolution structure of the b5 domain (PDB entry 3lf5) has previously been reported, which exhibits substantial differences in comparison to Cyb5A. The structural characterization of a construct comprising the naturally fused CS and b5R domains with bound FAD and NAD+ (PDB entry 6mv1) or NADP+ (PDB entry 6mv2) is now reported. The structures reveal that the linker between the CS and b5R cores is more ordered than predicted, with much of it extending the ß-sandwich motif of the CS domain. This limits the flexibility between the two domains, which recognize one another via a short ß-sheet motif and a network of conserved side-chain hydrogen bonds, salt bridges and cation-π interactions. Notable differences in FAD-protein interactions in Ncb5or and Cyb5R3 provide insight into the selectivity for docking of their respective b5 redox partners. The structures also afford a structural explanation for the unusual ability of Ncb5or to utilize both NADH and NADPH, and represent the first examples of native, fully oxidized b5R family members in which the nicotinamide ring of NAD(P)+ resides in the active site. Finally, the structures, together with sequence alignments, show that the b5R domain is more closely related to single-domain Cyb5R proteins from plants, fungi and some protists than to Cyb5R3 from animals.

19th International Meeting on Frankia and Actinorhizal Plants.

It has been 40 years since the first meeting dedicated to Frankia and actinorhizal plants, which was held at Petersham, Massachusetts (reported in Torrey and Tjepkema, 1979). Since then biennial meetings have been organised and held in different venues around the globe (Table 1). The most recent meeting, the "19th International Meeting on Frankia and Actinorhizal Plants", organised in Hammamet, Tunisia from 17th to 19th of March, 2018, gathered scientists from Algeria, Argentina, Belgium, China, Egypt, France, India, Portugal, Senegal, Sweden, UK, USA and Tunisia. The event was a stimulating opportunity for active researchers to share many advances since the previous meeting held in Montpellier, France (Franche et al. 2016) and to discuss new perspectives in this research field.

Single-Molecule FRET States, Conformational Interchange, and Conformational Selection by Dye Labels in Calmodulin.

We investigate the roles of measurement time scale and the nature of the fluorophores in the FRET states measured for calmodulin, a calcium signaling protein known to undergo pronounced conformational changes. The measured FRET distributions depend markedly on the measurement time scale (nanosecond or microsecond). Comparison of FRET distributions measured by donor fluorescence decay with FRET distributions recovered from single-molecule burst measurements binned over time scales of 90 µs to 1 ms reveals conformational averaging over the intervening time regimes. We find further that, particularly in the presence of saturating Ca(2+), the nature of the measured single-molecule FRET distribution depends markedly on the identity of the FRET pair. The results suggest interchange between conformational states on time scales of hundreds of microseconds or less. Interaction with a fluorophore such as the dye Texas Red alters both the nature of the measured FRET distributions and the dynamics of conformational interchange. The results further suggest that the fluorophore may not be merely a benign reporter of protein conformations in FRET studies, but may in fact alter the conformational landscape.

Heme uptake and metabolism in bacteria.

All but a few bacterial species have an absolute need for heme, and most are able to synthesize it via a pathway that is highly conserved among all life domains. Because heme is a rich source for iron, many pathogenic bacteria have also evolved processes for sequestering heme from their hosts. The heme biosynthesis pathways are well understood at the genetic and structural biology levels. In comparison, much less is known about the heme acquisition, trafficking, and degradation processes in bacteria. Gram-positive and Gram-negative bacteria have evolved similar strategies but different tactics for importing and degrading heme, likely as a consequence of their different cellular architectures. The differences are manifested in distinct structures for molecules that perform similar functions. Consequently, the aim of this chapter is to provide an overview of the structural biology of proteins and protein-protein interactions that enable Gram-positive and Gram-negative bacteria to sequester heme from the extracellular milieu, import it to the cytosol, and degrade it to mine iron.

The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese.

The history of cheese manufacture is a "natural history" in which animals, microorganisms, and the environment interact to yield human food. Part of the fascination with cheese, both scientifically and culturally, stems from its ability to assume amazingly diverse flavors as a result of seemingly small details in preparation. In this review, we trace the roots of cheesemaking and its development by a variety of human cultures over centuries. Traditional cheesemakers observed empirically that certain environments and processes produced the best cheeses, unwittingly selecting for microorganisms with the best biochemical properties for developing desirable aromas and textures. The focus of this review is on the role of fungi in cheese ripening, with a particular emphasis on the yeast-like fungus Geotrichum candidum. Conditions that encourage the growth of problematic fungi such as Mucor and Scopulariopsis as well as Arachnida (cheese mites), and how such contaminants might be avoided, are discussed. Bethlehem cheese, a pressed, uncooked, semihard, Saint-Nectaire-type cheese manufactured in the United Sates without commercial strains of bacteria or fungi, was used as a model for the study of stable microbial succession during ripening in a natural environment. The appearance of fungi during a 60-day ripening period was documented using light and scanning electron microscopy, and it was shown to be remarkably reproducible and parallel to the course of ripening of authentic Saint-Nectaire cheese in the Auvergne region of France. Geotrichum candidum, Mucor, and Trichothecium roseum predominate the microbiotas of both cheese types. Geotrichum in particular was shown to have high diversity in different traditional cheese ripening environments, suggesting that traditional manufacturing techniques selected for particular fungi. This and other studies suggest that strain diversity arises in relation to the lore and history of the regions from which these types of cheeses arose.

Growth and development of Frankia spp. strain CcI3 at the single-hypha level in liquid culture.

Filamentous actinobacteria from the genus Frankia grow by hyphal tip extension and branching. The growth kinetics and branching pattern of Frankia are not well studied, especially at the early stages of mycelial development. Here, we compare the growth of Frankia sp. strain CcI3 in liquid cultures with and without proteose peptone #3 (PP3) using time-lapse photomicrography and image analysis. Individual hyphae showed a pseudolinear increase in length at early stages of development, whereas at the mycelial level, the aggregate length of hyphae described an exponential rate before slowing. Growth based on optical density or microscopic observations was similar in medium with or without PP3. However, PP3 altered the pattern of mycelial development by increasing branching. Distances between the hyphal apex and first branches were on average shorter in PP3-containing media. The final interbranch distances were also shorter in PP3 medium indicating that hyphae tended to branch earlier and more often when supplemented with PP3 to give a more compact mycelium. Vesicle development in nitrogen-fixing cultures limited cell expansion as a result of vesicles truncating growth on new branches. The results provide some explanation for the growth kinetics of Frankia and some indication of how growth rates may be improved.

Genome sequence of "Candidatus Frankia datiscae" Dg1, the uncultured microsymbiont from nitrogen-fixing root nodules of the dicot Datisca glomerata.

Members of the noncultured clade of Frankia enter into root nodule symbioses with actinorhizal species from the orders Cucurbitales and Rosales. We report the genome sequence of a member of this clade originally from Pakistan but obtained from root nodules of the American plant Datisca glomerata without isolation in culture.

Transcriptomes of Frankia sp. strain CcI3 in growth transitions.

BACKGROUND: Frankia sp. strains are actinobacteria that form N2-fixing root nodules on angiosperms. Several reference genome sequences are available enabling transcriptome studies in Frankia sp. Genomes from Frankia sp. strains differ markedly in size, a consequence proposed to be associated with a high number of indigenous transposases, more than 200 of which are found in Frankia sp. strain CcI3 used in this study. Because Frankia exhibits a high degree of cell heterogeneity as a consequence of its mycelial growth pattern, its transcriptome is likely to be quite sensitive to culture age. This study focuses on the behavior of the Frankia sp. strain CcI3 transcriptome as a function of nitrogen source and culture age. RESULTS: To study global transcription in Frankia sp. CcI3 grown under different conditions, complete transcriptomes were determined using high throughput RNA deep sequencing. Samples varied by time (five days vs. three days) and by culture conditions (NH4+ added vs. N2 fixing). Assembly of millions of reads revealed more diversity of gene expression between five-day and three-day old cultures than between three day old cultures differing in nitrogen sources. Heat map analysis organized genes into groups that were expressed or repressed under the various conditions compared to median expression values. Twenty-one SNPs common to all three transcriptome samples were detected indicating culture heterogeneity in this slow-growing organism. Significantly higher expression of transposase ORFs was found in the five-day and N2-fixing cultures, suggesting that N starvation and culture aging provide conditions for on-going genome modification. Transposases have previously been proposed to participate in the creating the large number of gene duplication or deletion in host strains. Subsequent RT-qPCR experiments confirmed predicted elevated transposase expression levels indicated by the mRNA-seq data. CONCLUSIONS: The overall pattern of gene expression in aging cultures of CcI3 suggests significant cell heterogeneity even during normal growth on ammonia. The detection of abundant transcription of nif (nitrogen fixation) genes likely reflects the presence of anaerobic, N-depleted microsites in the growing mycelium of the culture, and the presence of significantly elevated transposase transcription during starvation indicates the continuing evolution of the Frankia sp. strain CcI3 genome, even in culture, especially under stressed conditions. These studies also sound a cautionary note when comparing the transcriptomes of Frankia grown in root nodules, where cell heterogeneity would be expected to be quite high.

The biology of Frankia sp. strains in the post-genome era.

Progress in understanding symbiotic determinants involved in the N(2)-fixing actinorhizal plant symbioses has been slow but steady. Problems persist with studying the bacterial contributions to the symbiosis using traditional microbiological techniques. However, recent years have seen the emergence of several genomes from Frankia sp. strains and the development of techniques for manipulating plant gene expression. Approaches to understanding the bacterial side of the symbiosis have employed a range of techniques that reveal the proteomes and transcriptomes from both cultured and symbiotic frankiae. The picture beginning to emerge provides some perspective on the heterogeneity of frankial populations in both conditions. In general, frankial populations in root nodules seem to maintain a rather robust metabolism that includes nitrogen fixation and substantial biosynthesis and energy-generating pathways, along with a modified ammonium assimilation program. To date, particular bacterial genes have not been implicated in root nodule formation but some hypotheses are emerging with regard to how the plant and microorganism manage to coexist. In particular, frankiae seem to present a nonpathogenic presence to the plant that may have the effect of minimizing some plant defense responses. Future studies using high-throughput approaches will likely clarify the range of bacterial responses to symbiosis that will need to be understood in light of the more rapidly advancing work on the plant host.

Accommodating a nonconservative internal mutation by water-mediated hydrogen bonding between ß-sheet strands: a comparison of human and rat type B (mitochondrial) cytochrome b5.

Mammalian type B (mitochondrial) b(5) cytochromes exhibit greater amino acid sequence diversity than their type A (microsomal) counterparts, as exemplified by the type B proteins from human (hCYB5B) and rat (rCYB5B). The comparison of X-ray crystal structures of hCYB5B and rCYB5B reported herein reveals a striking difference in packing involving the five-strand ß-sheet, which can be attributed to fully buried residue 21 in strand ß4. The greater bulk of Leu21 in hCYB5B in comparison to that of Thr21 in rCYB5B results in a substantial displacement of the first two residues in ß5, and consequent loss of two of the three hydrogen bonds between ß5 and ß4. Hydrogen bonding between the residues is instead mediated by two well-ordered, fully buried water molecules. In a 10 ns molecular dynamics simulation, one of the buried water molecules in the hCYB5B structure exchanged readily with solvent via intermediates having three water molecules sandwiched between ß4 and ß5. When the buried water molecules were removed prior to a second 10 ns simulation, ß4 and ß5 formed persistent hydrogen bonds identical to those in rCYB5B, but the Leu21 side chain was forced to adopt a rarely observed conformation. Despite the apparently greater ease of access of water to the interior of hCYB5B than of rCYB5B suggested by these observations, the two proteins exhibit virtually identical stability, dynamic, and redox properties. The results provide new insight into the factors stabilizing the cytochrome b(5) fold.

Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.

NADH cytochrome b(5) oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b(5) (b(5)), CHORD-SGT1 (CS), and cytochrome b(5) reductase (b(5)R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b(5) and b(5)R domains (Ncb5or-b(5) and Ncb5or-b(5)R, respectively) and compared them with human microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b(5) reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His(89) and His(112), consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b(5) family shown to have such a heme environment. Like other b(5) family members, Ncb5or-b(5) has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b(5) differs from Cyb5A with respect to location of the second heme ligand (His(112)) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b(5)R to Ncb5or-b(5) is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b(5) and b(5)R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b(5)R domains suggest that the CS domain facilitates docking of the b(5) and b(5)R domains. Trp(114) is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b(5)R domain to the b(5) domain.

Insertion sequence content reflects genome plasticity in strains of the root nodule actinobacterium Frankia.

BACKGROUND: Genome analysis of three Frankia sp. strains has revealed a high number of transposable elements in two of the strains. Twelve out of the 20 major families of bacterial Insertion Sequence (IS) elements are represented in the 148 annotated transposases of Frankia strain HFPCcI3 (CcI3) comprising 3% of its total coding sequences (CDS). EAN1pec (EAN) has 183 transposase ORFs from 13 IS families comprising 2.2% of its CDS. Strain ACN14a (ACN) differs significantly from the other strains with only 33 transposase ORFs (0.5% of the total CDS) from 9 IS families. RESULTS: Insertion sequences in the Frankia genomes were analyzed using BLAST searches, PHYML phylogenies and the IRF (Inverted Repeat Finder) algorithms. To identify putative or decaying IS elements, a PSI-TBLASTN search was performed on all three genomes, identifying 36%, 39% and 12% additional putative transposase ORFs than originally annotated in strains CcI3, EAN and ACN, respectively. The distribution of transposase ORFs in each strain was then analysed using a sliding window, revealing significant clustering of elements in regions of the EAN and CcI3 genomes. Lastly the three genomes were aligned with the MAUVE multiple genome alignment tool, revealing several Large Chromosome Rearrangement (LCR) events; many of which correlate to transposase clusters. CONCLUSION: Analysis of transposase ORFs in Frankia sp. revealed low inter-strain diversity of transposases, suggesting that the majority of transposase proliferation occurred without recent horizontal transfer of novel mobile elements from outside the genus. Exceptions to this include representatives from the IS3 family in strain EAN and seven IS4 transposases in all three strains that have a lower G+C content, suggesting recent horizontal transfer. The clustering of transposase ORFs near LCRs revealed a tendency for IS elements to be associated with regions of chromosome instability in the three strains. The results of this study suggest that IS elements may help drive chromosome differences in different Frankia sp. strains as they have adapted to a variety of hosts and environments.

On the nature of fur evolution: a phylogenetic approach in Actinobacteria.

BACKGROUND: An understanding of the evolution of global transcription regulators is essential for comprehending the complex networks of cellular metabolism that have developed among related organisms. The fur gene encodes one of those regulators - the ferric uptake regulator Fur - widely distributed among bacteria and known to regulate different genes committed to varied metabolic pathways. On the other hand, members of the Actinobacteria comprise an ecologically diverse group of bacteria able to inhabit various natural environments, and for which relatively little is currently understood concerning transcriptional regulation. RESULTS: BLAST analyses revealed the presence of more than one fur homologue in most members of the Actinobacteria whose genomes have been fully sequenced. We propose a model to explain the evolutionary history of fur within this well-known bacterial phylum: the postulated scenario includes one duplication event from a primitive regulator, which probably had a broad range of co-factors and DNA-binding sites. This duplication predated the appearance of the last common ancestor of the Actinobacteria, while six other duplications occurred later within specific groups of organisms, particularly in two genera: Frankia and Streptomyces. The resulting paralogues maintained main biochemical properties, but became specialised for regulating specific functions, coordinating different metal ions and binding to unique DNA sequences. The presence of syntenic regions surrounding the different fur orthologues supports the proposed model, as do the evolutionary distances and topology of phylogenetic trees built using both Neighbor-Joining and Maximum-Likelihood methods. CONCLUSION: The proposed fur evolutionary model, which includes one general duplication and two in-genus duplications followed by divergence and specialization, explains the presence and diversity of fur genes within the Actinobacteria. Although a few rare horizontal gene transfer events have been reported, the model is consistent with the view of gene duplication as a main force of microbial genomes evolution. The parallel study of Fur phylogeny across diverse organisms offers a solid base to guide functional studies and allows the comparison between response mechanisms in relation with the surrounding environment. The survey of regulators among related genomes provides a relevant tool for understanding the evolution of one of the first lines of cellular adaptability, control of DNA transcription.

The implication of life style on codon usage patterns and predicted highly expressed genes for three Frankia genomes.

Frankia are nitrogen-fixing actinomycetes that form a symbiotic association with over 200 species of woody dicotyledonous plants. Recently, three Frankia genomes were completely sequenced. In this study, the synonymous codon usage patterns of three Frankia genomes (strains CcI3, ACN14a, and EAN1pec) were determined and compared to each other and to other actinobacteria. As expected for a high G+C organism, codon usage by Frankia was highly biased, but differences were observed among the three strains. Using the codon adaptation index (CAI) as a numerical estimator of gene expression level, highly expressed genes in Frankia were predicted with ribosomal protein genes as a reference. The analysis of the predicted highly expressed genes showed that Frankia strain CcI3 had a different profile from the other two strains. Strain CcI3 had fewer predicted highly expressed genes in several COG categories including lipid transport and metabolism, secondary metabolites biosynthesis, inorganic ion transport and metabolism, and general function prediction only than Frankia strains EAN1pec and ACN14a. Interestingly, Frankia EAN1pec had more predicted highly expressed genes in transcription and signal transduction mechanisms than the other two strains. These differences were not just a reflection in total gene numbers, but also based on percentage of genes within a category. These results support the hypothesis that strain CcI3 is becoming a symbiotic specialist and the other two facultative symbiotic strains are maintaining their capacity to exist as free-living soil dwellers.

Comparative secretome analysis suggests low plant cell wall degrading capacity in Frankia symbionts.

BACKGROUND: Frankia sp. strains, the nitrogen-fixing facultative endosymbionts of actinorhizal plants, have long been proposed to secrete hydrolytic enzymes such as cellulases, pectinases, and proteases that may contribute to plant root penetration and formation of symbiotic root nodules. These or other secreted proteins might logically be involved in the as yet unknown molecular interactions between Frankia and their host plants. We compared the genome-based secretomes of three Frankia strains representing diverse host specificities. Signal peptide detection algorithms were used to predict the individual secretomes of each strain, and the set of secreted proteins shared among the strains, termed the core Frankia secretome. Proteins in the core secretome may be involved in the actinorhizal symbiosis. RESULTS: The Frankia genomes have conserved Sec (general secretory) and Tat (twin arginine translocase) secretion systems. The potential secretome of each Frankia strain comprised 4-5% of the total proteome, a lower percentage than that found in the genomes of other actinobacteria, legume endosymbionts, and plant pathogens. Hydrolytic enzymes made up only a small fraction of the total number of predicted secreted proteins in each strain. Surprisingly, polysaccharide-degrading enzymes were few in number, especially in strain CcI3, with more esterolytic, lipolytic and proteolytic enzymes having signal peptides. A total of 161 orthologous proteins belong to the core Frankia secretome. Of these, 52 also lack homologs in closely related actinobacteria, and are termed "Frankia-specific." The genes encoding these conserved secreted proteins are often clustered near secretion machinery genes. CONCLUSION: The predicted secretomes of Frankia sp. are relatively small and include few hydrolases, which could reflect adaptation to a symbiotic lifestyle. There are no well-conserved secreted polysaccharide-degrading enzymes present in all three Frankia genomes, suggesting that plant cell wall polysaccharide degradation may not be crucial to root infection, or that this degradation varies among strains. We hypothesize that the relative lack of secreted polysaccharide-degrading enzymes in Frankia reflects a strategy used by these bacteria to avoid eliciting host defense responses. The esterases, lipases, and proteases found in the core Frankia secretome might facilitate hyphal penetration through the cell wall, release carbon sources, or modify chemical signals. The core secretome also includes extracellular solute-binding proteins and Frankia-specific hypothetical proteins that may enable the actinorhizal symbiosis.

Enhancing the thermal stability of mitochondrial cytochrome b5 by introducing a structural motif characteristic of the less stable microsomal isoform.

Outer mitochondrial membrane cytochrome b5 (OM b5) is the most thermostable cytochrome b5 isoform presently known. Herein, we show that OM b5 thermal stability is substantially enhanced by swapping an apparently invariant motif in its heme-independent folding core with the corresponding motif characteristic of its less stable evolutionary relative, microsomal cytochrome b5 (Mc b5). The motif swap involved replacing two residues, Arg15 with His and Glu20 with Ser, thereby introducing a Glu11-His15-Ser20 H-bonding triad on the protein surface along with a His15/Trp22 pi-stacking interaction. The ferric and ferrous forms of the OM b5 R15H/E20S double mutant have thermal denaturation midpoints (Tm values) of approximately 93 degrees C and approximately 104 degrees C, respectively. A 15 degrees C increase in apoprotein Tm plays a key role in the holoprotein thermal stability enhancement, and is achieved by one of the most common natural mechanisms for stabilization of thermophilic versus mesophilic proteins: raising the unfolding free energy along the entire stability curve.

Comparison of cytochromes b5 from insects and vertebrates.

We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species.

Weak-field anions displace the histidine ligand in a synthetic heme peptide but not in N-acetylmicroperoxidase-8: possible role of heme geometry differences.

We have recently reported that aquo and thioether complexes of the ferric cytochrome c heme peptide N-acetylmicroperoxidase-8 (FeIII-1) exhibit greater low-spin character than do the corresponding complexes of a synthetic, water-soluble, monohistidine-ligated heme peptide (FeIII-2; Cowley, A. B.; Lukat-Rodgers, G. S.; Rodgers, K. R.; Benson, D. R. Biochemistry 2004, 43, 1656-1666). Herein we report results of studies showing that weak-field ligands bearing a full (fluoride, chloride, hydroxide) or partial (phenoxide, thiocyanate) negative charge on the coordinating atom trigger dissociation of the axial His ligand in FeIII-2 but not in FeIII-1. We attribute the greater sensitivity of His ligation in FeIII-1 to weak-field anionic ligands than to weak-field neutral ligands to the following phenomena: (1) anionic ligands pull FeIII further from the mean plane of a porphyrin than do neutral ligands, which will have the effect of straining the His-Fe bond in FeIII-2, and (2) heme in FeIII-2 is likely to undergo a modest doming distortion following anion binding that will render the His-ligated side of the porphyrin concave, thereby increasing porphyrin/ligand steric interactions. We propose that ruffling of the heme in FeIII-1 is an important factor contributing to its ability to resist His dissociation by weak-field anions. First, ruffling should allow His to more closely approach the porphyrin than is possible in FeIII-2, thereby reducing bond strain following anion binding. Second, the ruffling deformation in FeIII-1, which is enforced by the double covalent heme-peptide linkage, will almost certainly prevent significant porphyrin doming.