Daniela Coppola

Antarctic bacterial haemoglobin and its role in the protection against nitrogen reactive species.

In a cold and oxygen-rich environment such as Antarctica, mechanisms for the defence against reactive oxygen and nitrogen species are needed and represent important components in the evolutionary adaptations. In the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125, the presence of multiple genes encoding 2/2 haemoglobins and a flavohaemoglobin strongly suggests that these proteins fulfil important physiological roles, perhaps associated to the peculiar features of the Antarctic habitat. In this work, the putative role of Ph-2/2HbO, encoded by the PSHAa0030 gene, was investigated by in vivo and in vitro experiments in order to highlight its involvement in NO detoxification mechanisms. The PSHAa0030 gene was cloned and then over-expressed in a flavohaemoglobin-deficient mutant of Escherichia coli, unable to metabolise NO, and the resulting strain was studied analysing its growth properties and oxygen uptake in the presence of NO. We here demonstrate that Ph-2/2HbO protects growth and cellular respiration of the heterologous host from the toxic effect of NO-donors. Unlike in Mycobacterium tuberculosis 2/2 HbN, the deletion of the N-terminal extension of Ph-2/2HbO does not seem to reduce the NO scavenging activity, showing that the N-terminal extension is not a requirement for efficient NO detoxification. Moreover, the ferric form of Ph-2/2HbO was shown to catalyse peroxynitrite isomerisation in vitro, confirming its potential role in the scavenging of reactive nitrogen species. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

The Globins of Cold-Adapted Pseudoalteromonas haloplanktis TAC125: From the Structure to the Physiological Functions

Evolution allowed Antarctic microorganisms to grow successfully under extreme conditions (low temperature and high O2 content), through a variety of structural and physiological adjustments in their genomes and development of programmed responses to strong oxidative and nitrosative stress. The availability of genomic sequences from an increasing number of cold-adapted species is providing insights to understand the molecular mechanisms underlying crucial physiological processes in polar organisms. The genome of Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct truncated globins exhibiting the 2/2 α-helical fold. One of these globins has been extensively characterised by spectroscopic analysis, kinetic measurements and computer simulation. The results indicate unique adaptive structural properties that enhance the overall flexibility of the protein, so that the structure appears to be resistant to pressure-induced stress. Recent results on a genomic mutant strain highlight the involvement of the cold-adapted globin in the protection against the stress induced by high O2 concentration. Moreover, the protein was shown to catalyse peroxynitrite isomerisation in vitro. In this review, we first summarise how cold temperatures affect the physiology of microorganisms and focus on the molecular mechanisms of cold adaptation revealed by recent biochemical and genetic studies. Next, since only in a very few cases the physiological role of truncated globins has been demonstrated, we also discuss the structural and functional features of the cold-adapted globin in an attempt to put into perspective what has been learnt about these proteins and their potential role in the biology of cold-adapted microorganisms.

Molecular adaptations in haemoglobins of notothenioid fishes.

Since haemoglobins of all animal species have the same haem group, differences in their properties, including oxygen affinity, electrophoretic mobility and pH sensitivity, must result from the interaction of the prosthetic group with specific amino-acid residues in the primary structure. For this reason, fish globins have been the subject of extensive studies in recent years, not only for their structural characteristics, but also because they offer the possibility to investigate the evolutionary history of these ancient molecules in marine and freshwater species living in a great variety of environmental conditions. This review summarizes the current knowledge on the structure, function and phylogeny of haemoglobins of notothenioid fishes. On the basis of crystallographic analysis, the evolution of the Root effect is analysed. Adaptation of the oxygen transport system in notothenioids seems to be based on evolutionary changes, involving levels of biological organization higher than the structure of haemoglobin. These include changes in the rate of haemoglobin synthesis or in regulation by allosteric effectors, which affect the amount of oxygen transported in blood. These factors are thought to be more important for short-term response to environmental challenges than previously believed.

ATP regulation of the ligand-binding properties in temperate and cold-adapted haemoglobins. X-ray structure and ligand-binding kinetics in the sub-Antarctic fish Eleginops maclovinus.

The major haemoglobin of the sub-Antarctic fish Eleginops maclovinus was structurally and functionally characterised with the aim to compare molecular environmental adaptations in the O(2)-transport system of sub-Antarctic fishes of the suborder Notothenioidei with those of their high-latitude relatives. Ligand-binding kinetics of the major haemoglobin of E. maclovinus indicated strong stabilisation of the liganded quaternary T state, enhanced in the presence of the physiological allosteric effector ATP, compared to that of high-Antarctic Trematomus bernacchii. The activation enthalpy for O(2) dissociation was dramatically lower than that in T. bernacchii haemoglobin, suggesting remarkable differences in temperature sensitivity and structural changes associated with O(2) release and exit from the protein. The haemoglobin functional properties, together with the X-ray structure of the CO form at 1.49 Å resolution, the first of a temperate notothenioid, strongly support the hypothesis that in E. maclovinus, whose life-style varies according to changes in habitat, the mechanisms that regulate O(2) affinity and the ATP-induced Root effect differ from those of high-Antarctic Notothenioids.

Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes

BackgroundConjugation of human and animal hemoglobins with polyethylene glycol has been widely explored as a means to develop blood substitutes, a novel pharmaceutical class to be used in surgery or emergency medicine. However, PEGylation of human hemoglobin led to products with significantly different oxygen binding properties with respect to the unmodified tetramer and high NO dioxygenase reactivity, known causes of toxicity. These recent findings call for the biotechnological development of stable, low-affinity PEGylated hemoglobins with low NO dioxygenase reactivity.ResultsTo investigate the effects of PEGylation on protein structure and function, we compared the PEGylation products of human hemoglobin and Trematomus bernacchii hemoglobin, a natural variant endowed with a remarkably low oxygen affinity and high tetramer stability. We show that extension arm facilitated PEGylation chemistry based on the reaction of T. bernacchii hemoglobin with 2-iminothiolane and maleimido-functionalyzed polyethylene glycol (MW 5000 Da) leads to a tetraPEGylated product, more homogeneous than the corresponding derivative of human hemoglobin. PEGylated T. bernacchii hemoglobin largely retains the low affinity of the unmodified tetramer, with a p50 50 times higher than PEGylated human hemoglobin. Moreover, it is still sensitive to protons and the allosteric effector ATP, indicating the retention of allosteric regulation. It is also 10-fold less reactive towards nitrogen monoxide than PEGylated human hemoglobin.ConclusionsThese results indicate that PEGylated hemoglobins, provided that a suitable starting hemoglobin variant is chosen, can cover a wide range of oxygen-binding properties, potentially meeting the functional requirements of blood substitutes in terms of oxygen affinity, tetramer stability and NO dioxygenase reactivity.

‘Cool’ adaptations to cold environments: globins in Notothenioidei (Actynopterygii, Perciformes)

Notothenioidei, the taxonomic group of teleosts that dominates the Southern Ocean and dwell in the Ross Sea at large, provide an example of marine species that underwent unique adaptations to life at low temperatures and high oxygen concentrations, resulting in morphological, physiological, genomic, and biochemical peculiarities in comparison with warm-water fish. Global Warming raises concerns over the fate of these stenothermal fish, as their adaptation has been accompanied by irreversible genomic losses, which suggest a poor genetic potential to adapt to warmer climates. Specifically, this review focuses on adaptation of proteins belonging to the globin superfamily, which include the respiratory proteins hemoglobin and myoglobin and the non-respiratory proteins neuroglobin and cytoglobin. Here, we describe their molecular adaptations to cold temperatures in the framework of the physiology of oxygen transport and management of oxidative stress in fish species largely populating the Ross Sea.

Marine Microbial Secondary Metabolites: Pathways, Evolution and Physiological Roles

Microbes produce a huge array of secondary metabolites endowed with important ecological functions. These molecules, which can be catalogued as natural products, have long been exploited in medical fields as antibiotics, anticancer and anti-infective agents. Recent years have seen considerable advances in elucidating natural-product biosynthesis and many drugs used today are natural products or natural-product derivatives. The major contribution to recent knowledge came from application of genomics to secondary metabolism and was facilitated by all relevant genes being organised in a contiguous DNA segment known as gene cluster. Clustering of genes regulating biosynthesis in bacteria is virtually universal. Modular gene clusters can be mixed and matched during evolution to generate structural diversity in natural products. Biosynthesis of many natural products requires the participation of complex molecular machines known as polyketide synthases and non-ribosomal peptide synthetases. Discovery of new evolutionary links between the polyketide synthase and fatty acid synthase pathways may help to understand the selective advantages that led to evolution of secondary-metabolite biosynthesis within bacteria. Secondary metabolites confer selective advantages, either as antibiotics or by providing a chemical language that allows communication among species, with other organisms and their environment. Herewith, we discuss these aspects focusing on the most clinically relevant bioactive molecules, the thiotemplated modular systems that include polyketide synthases, non-ribosomal peptide synthetases and fatty acid synthases. We begin by describing the evolutionary and physiological role of marine natural products, their structural/functional features, mechanisms of action and biosynthesis, then turn to genomic and metagenomic approaches, highlighting how the growing body of information on microbial natural products can be used to address fundamental problems in environmental evolution and biotechnology.

Marine Microbial Secondary Metabolites

Microbes produce a huge array of secondary metabolites endowed with important ecological functions. These molecules, which can be catalogued as natural products, have long been exploited in medical fields as antibiotics, anticancer and anti-infective agents. Recent years have seen considerable advances in elucidating natural-product biosynthesis and many drugs used today are natural products or natural-product derivatives. The major contribution to recent knowledge came from application of genomics to secondary metabolism and was facilitated by all relevant genes being organised in a contiguous DNA segment known as gene cluster. Clustering of genes regulating biosynthesis in bacteria is virtually universal. Modular gene clusters can be mixed and matched during evolution to generate structural diversity in natural products. Biosynthesis of many natural products requires the participation of complex molecular machines known as polyketide synthases and non-ribosomal peptide synthetases. Discovery of new evolutionary links between the polyketide synthase and fatty acid synthase pathways may help to understand the selective advantages that led to evolution of secondary-metabolite biosynthesis within bacteria. Secondary metabolites confer selective advantages, either as antibiotics or by providing a chemical language that allows communication among species, with other organisms and their environment. Herewith, we discuss these aspects focusing on the most clinically relevant bioactive molecules, the thiotemplated modular systems that include polyketide synthases, non-ribosomal peptide synthetases and fatty acid synthases. We begin by describing the evolutionary and physiological role of marine natural products, their structural/functional features, mechanisms of action and biosynthesis, then turn to genomic and metagenomic approaches, highlighting how the growing body of information on microbial natural products can be used to address fundamental problems in environmental evolution and biotechnology.

Coexistence of multiple globin genes conferring protection against nitrosative stress to the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Despite the large number of globins recently discovered in bacteria, our knowledge of their physiological functions is restricted to only a few examples. In the microbial world, globins appear to perform multiple roles in addition to the reversible binding of oxygen; all these functions are attributable to the heme pocket that dominates functional properties. Resistance to nitrosative stress and involvement in oxygen chemistry seem to be the most prevalent functions for bacterial globins, although the number of globins for which functional roles have been studied via mutation and genetic complementation is very limited. The acquisition of structural information has considerably outpaced the physiological and molecular characterisation of these proteins. The genome of the Antarctic cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) contains genes encoding three distinct single-chain 2/2 globins, supporting the hypothesis of their crucial involvement in a number of functions, including protection against oxidative and nitrosative stress in the cold and O2-rich environment. In the genome of PhTAC125, the genes encoding 2/2 globins are constitutively transcribed, thus suggesting that these globins are not functionally redundant in their physiological function in PhTAC125. In the present study, the physiological role of one of the 2/2 globins, Ph-2/2HbO-2217, was investigated by integrating in vivo and in vitro results. This role includes the involvement in the detoxification of reactive nitrogen and O2 species including NO by developing two in vivo and in vitro models to highlight the protective role of Ph-2/2HbO-2217 against reactive nitrogen species. The PSHAa2217 gene was cloned and over-expressed in the flavohemoglobin-deficient mutant of Escherichia coli and the growth properties and O2 uptake in the presence of NO of the mutant carrying the PSHAa2217 gene were analysed. The ferric form of Ph-2/2HbO-2217 is able to catalyse peroxynitrite isomerisation in vitro, indicating its potential role in the scavenging of reactive nitrogen species. Here we present in vitro evidence for the detoxification of NO by Ph-2/2HbO-2217.

Functional characterisation of the haemoglobins of the migratory notothenioid fish Dissostichus eleginoides

This study addresses the primary structure, the oxygen-binding properties and the CO-rebinding kinetics of the haemoglobins of the Patagonian toothfish Dissostichus eleginoides. D. eleginoides belongs to the family Nototheniidae, the most diversified of the suborder Notothenioidei, mostly exhibiting an Antarctic distribution. Some of its features are typical of Antarctic species, some are not. For instance, D. eleginoides appears not to have functional antifreeze glycoproteins (consistent with its non-Antarctic distribution). In contrast, it has a major and a minor haemoglobin (similar to many Antarctic notothenioids), and their very low oxygen affinity does not follow the trend of other non-Antarctic notothenioids and appears typical of cold-adapted species. Moreover, the amino-acid sequence reveals high identity with the globins of Antarctic notothenioids, arguing in favour of a common origin within notothenioids, and indicates that the primary structure of the major and minor haemoglobins has undergone modifications only to a limited extent. The ligand-rebinding kinetics of the major haemoglobin of D. eleginoides indicate a strong stabilisation of the quaternary T state at lower pH values.