Structural analysis of protein–protein interactions in type I polyketide synthases

Abstract

Polyketide synthases (PKSs) are responsible for synthesizing a myriad of natural products with agricultural, medicinal relevance. The PKSs consist of multiple functional domains of which each can catalyze a specified chemical reaction leading to the synthesis of polyketides. Biochemical studies showed that protein–substrate and protein–protein interactions play crucial roles in these complex regio-/stereo-selective biochemical processes. Recent developments on X-ray crystallography and protein NMR techniques have allowed us to understand the biosynthetic mechanism of these enzymes from their structures. These structural studies have facilitated the elucidation of the sequence–function relationship of PKSs and will ultimately contribute to the prediction of product structure. This review will focus on the current knowledge of type I PKS structures and the protein–protein interactions in this system.     

Gibberellin Biosynthesis in Plants and Fungi: A Case of Convergent Evolution?

As well as being phytohormones, gibberellins (GAs) are present in some fungi and bacteria. Indeed, GAs were first discovered in the fungus Gibberella fujikuroi, from which gibberellic acid (GA3) and other GAs are produced commercially. Although higher plants and the fungus produce structurally identical GAs, there are important differences in the pathways and enzymes involved. This has become particularly apparent with the identification of almost all of the genes for GA-biosynthesis in Arabidopsis thaliana and G. fujikuroi, following the sequencing of the Arabidopsis genome and the detection of a GA-biosynthesis gene cluster in the fungus. For example, 3b-hydroxylation occurs early in the pathway in G. fujikuroi and is catalyzed by a cytochrome P450 monooxygenase, whereas it is usually the final step in plants and is catalyzed by 2-oxoglutarate-dependent dioxygenases. Similarly, 20-oxidation is catalyzed by dioxygenases in plants and a cytochrome P450 in the fungus. Even where cytochrome P450s have equivalent functions in plants and Gibberella, they are unrelated in terms of amino acid sequence. These profound differences indicate that higher plants and fungi have evolved their complex biosynthetic pathways to GAs independently and not by horizontal gene transfer.     

Polyketide synthases of bacterial symbionts in sponges – Evolution-based applications in natural products research

Marine sponges are an unusually rich source of bioactive natural products with clinical potential. They also often harbor rich communities of symbiotic bacteria that have often been suspected as the true producers of sponge-derived compounds. To date, these bacteria can in most cases not be cultivated, but culture-independent methods, such as isolating and analyzing biosynthetic gene clusters using metagenomic strategies, have recently provided first insights into their chemical potential. This review summarizes recent work of our laboratory on the study of polyketide synthases (PKSs). These studies revealed two evolutionarily distinct, unusual PKS types that are commonly found in sponge metagenomes and were shown to be of bacterial origin. One, the sup PKS, dominates sponge metagenomic DNA libraries, occurs widespread in bacteriosponges and is to date exclusively known from such animals. Data suggest that it is a type of synthase that generates methyl-branched fatty acids, which are commonly present in sponges. The other PKS type, termed trans-acyltransferase (AT) PKS, is responsible for the biosynthesis of complex, bioactive polyketides, such as the onnamides, and also occurs in free-living bacteria. The diversity of PKS genes present in a single sponge metagenome can be enormous. However, the phylogenetic approaches outlined in this review can provide valuable insights into the PKS function and structures of polyketides and can assist in the targeted isolation of gene clusters.     

Evolution of acyl-ACP thioesterases and β-ketoacyl-ACP synthases revealed by protein–protein interactions

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein–protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners, whereas KSs showed promiscuous behavior across bacterial, plant, and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic cross-linking, and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes.     

Biosynthesis of plastoquinone and β-carotene in isolated chloroplasts

Intact, isolated spinach chloroplasts incorporated ¹⁴C from ¹⁴CO₂ into plastoquinone and β-carotene under photosynthetic conditions. Addition of unlabelled l-tyrosine, p-hydroxyphenylpyruvate, or homogentisate increased the incorporation of ¹⁴C into plastoquinone, but decreased that into β-carotene.     

Evolution and functional diversification of the small heat shock protein/α-crystallin family in higher plants

Small heat shock proteins (sHSPs) are chaperones that play an important role in stress tolerance. They consist of an alpha-crystallin domain (ACD) flanked by N- and C-terminal regions. However, not all proteins that contain an ACD, hereafter referred to as ACD proteins, are sHSPs because certain ACD proteins are known to have different functions. Furthermore, since not all ACD proteins have been identified yet, current classifications are incomplete. A total of 17 complete plant proteomes were screened for the presence of ACD proteins by HMMER profiling and the identified ACD protein sequences were classified by maximum likelihood phylogeny. Differences among and within groups were analysed, and levels of functional constraint were determined. There are 29 different classes of ACD proteins, eight of which contain classical sHSPs and five likely chaperones. The other classes contain proteins with uncharacterised or poorly characterised functions. N- and C-terminal sequences are conserved within the phylogenetic classes. Phylogenetics suggests a single duplication of the CI sHSP ancestor that occurred prior to the speciation of mono- and dicotyledons. This was followed by a number of more recent duplications that resulted in the presence of many paralogues. The results suggest that N- and C-terminal sequences of sHSPs play a role in class-specific functionality and that non-sHSP ACD proteins have conserved but unexplored functions, which are mainly determined by subsequences other than that of the ACD.     

Evolution of Dengue Disease and Entomological Monitoring in Santa Cruz,Bolivia 2002 – 2008

BackgroundIn the context of a rapid increase of dengue cases in the Americas, a monitoring system based on systematic serological control (IgM) of patients consulting for suspected dengue was developed in Bolivia at the end of the 1990s. In the most affected city of Santa Cruz, this system was complemented by an entomological surveillance program based on periodical search for immature stages of Aedes aegypti in dwelling water-holding containers. Here, we analyze these data and describe dengue patterns over 6 years (2002–2008), highlighting the spatial distribution of patients and vectors.Conclusions/SignificanceOver the years, the increasing trend of dengue cases has been highlighted as well as its widespread distribution over the entire city, but an underestimation of the number of cases is strongly suspected. Contrary to popular belief, the city center appears more affected than the periphery, and dengue is not particularly related to waste. Interestingly, the clinical diagnosis of dengue by physicians improved over the years, whatever the gender, age and residential area of suspected cases.     

CLO-PLA2 – a database of clonal plants in central Europe

CLO-PLA2' (CLOnal PLAnts, version 2) is a database on architectural aspects of clonal growth in vascular plants of central Europe. The database includes 2749 species, characterised by 25 variables, either directly or indirectly related to clonal growth. The total number of items in the database is over 12750. The structure of the database is described and the variables used to characterise clonal growth of individual species are listed. Two examples of database utilisation are given. The first concerns the relationship between habitat niche width and the mode of clonal growth. Turf graminoids, species with long-lived rhizomes either short to long and formed below-ground, or short and formed above-ground, and short-lived rhizomes formed above-ground, are over-represented among the species with very broad niches and under-represented among the species with narrow niches. In contrast, species multiplying by plant fragments are missing among the species with the broadest niches. The second example explores how individual clonal growth modes are combined in individual species. About 21% of species of clonal plants have more than one mode of clonal growth. Some combinations are over-represented in certain families and environments. The application of phylogenetic independent contrasts (PIC) showed that both phylogenetic constraints and adaptations to particular environmental conditions play important roles in determining the observed pattern.     

Biosynthesis and degradation of α-tomatine in developing tomato fruits

Fruits of tomato incorporated [2-¹⁴C]mevalonic acid lactone into the steroidal glycoalkaloid α-tomatine. Young fruits showed the greatest alkaloid-synthesizing ability but this decreased as the fruits developed. Analysis of sap exuded from fruit stalks and also application of[4-¹⁴C]cholesterol to leaves confirmed that tomatine is not transported into fruits from vegetative organs. Accumulation of this alkaloid in fruits thus appears entirely due to synthesis. Excised fruits of all developmental stages degraded injected [¹⁴C]tomatine and rates were directly related to fruit age. The pattern of accumulation/decline in fruit tomatine may be explicable on the basis of changing capacity for synthesis/degradation during development. Label from injected [¹⁴C]tomatine was present mainly in chlorophylls and carotenoids where it increased with time as that in tomatine decreased. The significance of the relationship between tomatine disappearance and carotenoid development is briefly discussed. The aglycone tomatidine was not detected in green fruits but a Δ¹⁶-5α-pregnenolone-like compound was.     

Characterization of haemoglobin from Actinorhizal plants – An in silico approach

Plant haemoglobins (Hbs), found in both symbiotic and non-symbiotic plants, are heme proteins and members of the globin superfamily. Hb genes of actinorhizal Fagales mostly belong to the non-symbiotic type of haemoglobin; however, along with the non-symbiotic Hb, Casuarina sp. posses a symbiotic one (symCgHb), which is expressed specifically in infected cells of nodules. A thorough sequence analysis of 26 plant Hb proteins, currently available in public domain, revealed a consensus motif of 29 amino acids. This motif is present in all the members of symbiotic class II Hbs including symCgHb and non-symbiotic Class II Hbs, but is totally absent in Class I symbiotic and non-symbiotic Hbs. Further, we constructed 3D structures of Hb proteins from Alnus and Casuarina through homology modelling and peeped into their structural properties. Structure-based studies revealed that the Casuarina symbiotic haemoglobin protein shows distinct stereochemical properties from that of the other Casuarina and Alnus Hb proteins. It also showed considerable structural similarities with leghemoglobin structure from yellow lupin (pdb id 1GDI). Therefore, sequence and structure analyses point to the fact that symCgHb protein shows significant resemblance to symbiotic haemoglobin found in legumes and may thus eventually play a similar role in shielding the nitrogenase from oxygen as seen in the case of leghemoglobin.     

Cytokinin metabolism in Physcomitrella patens – differences and similarities to higher plants

Cytokinins play an important role in plant development and occur informs with different hormonal activity. As the nucleotide forms of cytokininsare considered to have little or no biological activity, the conversion ofcytokinin bases and ribosides to their nucleotides can contribute to the tuningof cytokinin activity in plant cells. Cytokinin metabolism was monitoredin vivo by feeding either radiolabelledisopentenyladenosine (³H-[9R]iP) or isopentenyladenine(³H-iP) to liquid grown chloronema tissue ofPhyscomitrellapatens (Hedw.) B.S.G. wild type. The riboside ³H-[9R]iPwas rapidly converted to ³H-iP, which was released into the culturemedium. The intracellular concentration of the ³H-iP was twice ashigh as extracellular. From the overall amount of ³H-iP about 95%were present in the medium. Cytokinin nucleotides occurred as tritiated mono-,di- and triphosphates of ³H-[9R]iP. When feeding the base³H-iP however, its main metabolic fate was degradation and nosignificant amounts of radiolabelled cytokinin nucleotides were detected. Forthe cytokinin metabolism in P. patens it is concludedthat,in contrast to higher plants nucleotides are mainly formed from ribosidesvia the adenosine kinase pathway and not byribophosphorylation of the cytokinin base via adeninephosphoribosyltransferase.     

Involvement of Nitrogen-Containing Compounds in β -Lactam Biosynthesis and its Control

ABSTRACT

Biosynthesis of β -lactam antibiotics by fungi and actinomycetes is markedly affected by compounds containing nitrogen. The different processes employed by the spectrum of microbes capable of making these valuable compounds are affected differently by particular compounds. Ammonium ions, except at very low concentrations, exert negative effects via nitrogen metabolite repression, sometimes involving the nitrogen regulatory gene nre. Certain amino acids are precursors or inducers, whereas others are involved in repression and, in certain cases, as inhibitors of biosynthetic enzymes and of enzymes supplying precursors. The most important amino acids from the viewpoint of regulation are lysine, methionine, glutamate and valine. Surprisingly, diamines such as diaminopropane, putrescine and cadaverine induce cephamycin production by actinomycetes. In addition to penicillins and cephalosporins made by fungi and cephamycins made by actinomycetes, other β-lactams are made by actinomycetes and unicellular bacteria. These include clavams (e.g., clavulanic acid), carbapenems (e.g., thienamycin), nocardicins and monobactams. Here also, amino acids are precursors and inhibitors, but only little is known about regulation. In the case of the simplest carbapenem made by unicellular bacteria, i.e., 1-carba-2-em-3-carboxylic acid, quorum sensors containing homoserine lactone are inducers.     

Association of metabolites reflecting type III and VI collagen formation with modified Rodnan skin score in systemic sclerosis – a cross-sectional study

Objective: The objective was to investigate blood-based biomarkers of type I (PRO-C1), III (PRO-C3) and VI (PRO-C6) collagen formation in systemic sclerosis (SSc) patients and examine their correlation to modified Rodnan skin score (mRSS).

Methods: Limited (lSSc, n?=?76) and diffuse SSc (dSSc, n?=?41) fulfilling the ACR/EULAR 1980 and 2013 classification criteria for SSc and asymptomatic controls (n?=?9) were included. PRO-C1, PRO-C3 and PRO-C6 were measured in serum.

Results: LSSc compared to dSSc were significantly older, had longer disease duration and lower mRSS. PRO-C3 was higher in early dSSc compared to early lSSc (mean [95 percentile], 27.4 [13.1–39.1] ng/mL vs 14.9 [8.2–28.8] ng/mL, p?=?0.006). PRO-C6 levels were higher in early dSSc compared to early lSSc and late dSSc (early dSSc: 28.2 [10.4–92.3] ng/ml vs early lSSc: 11.0 [6.9–28.5] ng/ml; p?=?0.006 and late dSSc: 12.6 [6.5–25.3] ng/mL, p?=?0.04). No difference was observed with PRO-C1. PRO-C3 and PRO-C6 were moderately correlated with mRSS with R-partials of 0.36 (p?<?0.001) and 0.29 (p?=?0.002), respectively

Conclusion: Measures of type III and VI collagen formation are potential objective biomarkers of fibrosis in systemic sclerosis. These biomarkers could be useful in monitoring the disease and efficacy of treatment.     

Polychlorinated biphenyls (pcbs) in fish-eating sea birds—III. Molecular features and metabolic interpretations of PCB isomers and congeners in adipose tissues

1. Enrichment factors have been calculated for several persistent PCB congeners in the adipose tissue for five species of fish-eating sea birds (female razorbills, puffins, guillemots, shags and cormorants) obtained from the same sites during 1978–1984 (see preceding papers).2. The enrichment factor of an individual PCB is expressed as its concentration in the tissue compared with its abundance in commerical mixtures of PCBs or compared with the concentration in the tissue of the abundant congener 2,2',4,4',5,5'-hexachlorobiphenyl (congener 153, IUPAC system of numbering).3. There were no significant differences between the five species in the enrichment factor of individual persistent PCBs compared with congener 153, indicating similar levels of diminished metabolism of this group of congeners.4. Of the 47 individual PCBs identified, ten congeners had enrichment factors of > 1 in all of the species and these accounted for up to 70% of the concentration of total PCBs present. Some of these persistent congeners had approximately coplanar configurations (i.e. non-ortho -substituted congeners). Five congeners, which accounted for about 35% of the total concentration of PCBs in the tissues, shared the molecular feature of chlorine substituents at adjacent meta-para carbon atoms.5. A number of congeners were identified with enrichment factors of <1 compared with their abundance in Aroclor 1260, and very striking differences were observed between the five species in the ratio of non-persistent congeners to the persistent congener 153. These non-persistent congeners share the molecular feature of at least one pair of adjacent unsubstituted meta-para carbon atoms in the rings. This agrees with our molecular “rule” (see preceding papers) that congeners with this structural feature are subjected to metabolism by the cytochrome P-450 component of hepatic microsomal monooxygenases.6. Evidence is presented that this molecular rule applies to the persistence or non-persistence of classes of PCBs in other biological systems and that the complete absence of H atoms at adjacent carbon atoms is an essential structural requirement for the accumulation of PCBs in tissues.7. The persistence or non-persistence of individual PCBs is compared with their ability to induce specific isoforms of the cytochrome P-450 components of hepatic microsomal monooxygenases, and the toxic effects of individual PCBs that accumulate is discussed in terms of the potential environmental hazard that they represent.     

Mineral Induced Phosphorylation of Glycolate Ion – a Metaphor in Chemical Evolution

Bilateral surface-active minerals with excess positive charge concentrate glycolate and trimetaphosphate ion from 10^–3 m aqueous solution to half-saturation of the internal surface sites, and induce phosphorylation of glycolate ion in the mineral with trimetaphosphate, sorbed from 10^–2 m solution. By utilizing reactants from dilute solution at near-neutral pH, and eliminating the need for participating organic nitrogen compounds, the reaction comprises several elements considered necessary for geochemical realism in models for molecular evolution.