Cloning and characterization of the Type I Baeyer-Villiger monooxygenase from Leptospira biflexa.

Baeyer-Villiger monooxygenases are recognized by their ability and high selectivity as oxidative biocatalysts for the generation of esters or lactones using ketones as starting materials. These enzymes represent valuable tools for biooxidative syntheses since they can catalyze reactions that otherwise involve strong oxidative reagents. In this work, we present a novel enzyme, the Type I Baeyer-Villiger monooxygenase from Leptospira biflexa. This protein is phylogenetically distant from other well-characterized BVMOs. In order to study this new enzyme, we cloned its gene, expressed it in Escherichia coli and characterized the substrate scope of the Baeyer-Villiger monooxygenase from L. biflexa as a whole-cell biocatalyst. For this purpose, we performed the screening of a collection of ketones with variable structures and sizes, namely acyclic ketones, aromatic ketones, cyclic ketones, and fused ketones. As a result, we observed that this biocatalyst readily oxidized linear- and branched- medium-chain ketones, alkyl levulinates and linear ketones with aromatic substituents with excellent regioselectivity. In addition, this enzyme catalyzed the oxidation of 2-substituted cycloketone derivatives but showed an unusual selection against substituents in positions 3 or 4 of the ring.

Environmental selection pressures related to iron utilization are involved in the loss of the flavodoxin gene from the plant genome.

Oxidative stress and iron limitation represent the grim side of life in an oxygen-rich atmosphere. The versatile electron transfer shuttle ferredoxin, an iron-sulfur protein, is particularly sensitive to these hardships, and its downregulation under adverse conditions severely compromises survival of phototrophs. Replacement of ferredoxin by a stress-resistant isofunctional carrier, flavin-containing flavodoxin, is a widespread strategy employed by photosynthetic microorganisms to overcome environmental adversities. The flavodoxin gene was lost in the course of plant evolution, but its reintroduction in transgenic plants confers increased tolerance to environmental stress and iron starvation, raising the question as to why a genetic asset with obvious adaptive value was not kept by natural selection. Phylogenetic analyses reveal that the evolutionary history of flavodoxin is intricate, with several horizontal gene transfer events between distant organisms, including Eukarya, Bacteria, and Archaea. The flavodoxin gene is unevenly distributed in most algal lineages, with flavodoxin-containing species being overrepresented in iron-limited regions and scarce or absent in iron-rich environments. Evaluation of cyanobacterial genomic and metagenomic data yielded essentially the same results, indicating that there was little selection pressure to retain flavodoxin in iron-rich coastal/freshwater phototrophs. Our results show a highly dynamic evolution pattern of flavodoxin tightly connected to the bioavailability of iron. Evidence presented here also indicates that the high concentration of iron in coastal and freshwater habitats may have facilitated the loss of flavodoxin in the freshwater ancestor of modern plants during the transition of photosynthetic organisms from the open oceans to the firm land.

Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications.

External flavoprotein monooxygenases comprise a group of flavin-dependent oxidoreductases that catalyze the insertion of one atom of molecular oxygen into an organic substrate and the second atom is reduced to water. These enzymes are involved in a great number of metabolic pathways both in prokaryotes and eukaryotes. Flavoprotein monooxygenases have attracted the attention of researchers for several decades and the advent of recombinant DNA technology caused a great progress in the field. These enzymes are subjected to detailed biochemical and structural characterization and some of them are also regarded as appealing oxidative biocatalysts for the production of fine chemicals and valuable intermediates toward active pharmaceutical ingredients due to their high chemo-, stereo-, and regioselectivity. Here, we review the most representative reactions catalyzed both in vivo and in vitro by prototype flavoprotein monooxygenases, highlighting the strategies employed to produce them recombinantly, to enhance the yield of soluble proteins, and to improve cofactor regeneration in order to obtain versatile biocatalysts. Although we describe the most outstanding features of flavoprotein monooxygenases, we mainly focus on enzymes that were cloned, expressed and used for biocatalysis during the last years.

Expression of the minor isoform pea ferredoxin in tobacco alters photosynthetic electron partitioning and enhances cyclic electron flow.

Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms.

The importance of flavodoxin for environmental stress tolerance in photosynthetic microorganisms and transgenic plants. Mechanism, evolution and biotechnological potential.

Ferredoxins are electron shuttles harboring iron-sulfur clusters which participate in oxido-reductive pathways in organisms displaying very different lifestyles. Ferredoxin levels decline in plants and cyanobacteria exposed to environmental stress and iron starvation. Flavodoxin is an isofunctional flavoprotein present in cyanobacteria and algae (not plants) which is induced and replaces ferredoxin under stress. Expression of a chloroplast-targeted flavodoxin in plants confers tolerance to multiple stresses and iron deficit. We discuss herein the bases for functional equivalence between the two proteins, the reasons for ferredoxin conservation despite its susceptibility to aerobic stress and for the loss of flavodoxin as an adaptive trait in higher eukaryotes. We also propose a mechanism to explain the tolerance conferred by flavodoxin when expressed in plants.

Flavodoxin displays dose-dependent effects on photosynthesis and stress tolerance when expressed in transgenic tobacco plants.

Ferredoxins are iron-sulfur proteins involved in various one-electron transfer pathways. Ferredoxin levels decrease under adverse environmental conditions in photosynthetic organisms. In cyanobacteria, this decline is compensated by induction of flavodoxin, an isofunctional flavoprotein. Flavodoxin is not present in higher plants, but transgenic Nicotiana tabacum lines accumulating Anabaena flavodoxin in plastids display increased tolerance to different sources of environmental stress. As the degree of tolerance correlated with flavodoxin dosage in plastids of nuclear-transformed transgenic tobacco, we prepared plants expressing even higher levels of flavodoxin by direct plastid transformation. A suite of nuclear- and chloroplast-transformed lines expressing a wide range of flavodoxin levels, from 0.3 to 10.8 µmol m(-2), did not exhibit any detectable growth phenotype relative to the wild type. In the absence of stress, the contents of both chlorophyll a and carotenoids, as well as the photosynthetic performance (photosystem II maximum efficiency, photosystem II operating efficiency, electron transport rates and carbon assimilation rates), displayed a moderate increase with flavodoxin concentrations up to 1.3-2.6 µmol flavodoxin m(-2), and then declined to wild-type levels. Stress tolerance, as estimated by the damage inflicted on exposure to the pro-oxidant methyl viologen, also exhibited a bell-shaped response, with a significant, dose-dependent increase in tolerance followed by a drop in the high-expressing lines. The results indicate that optimal photosynthetic performance and stress tolerance were observed at flavodoxin levels comparable to those of endogenous ferredoxin. Further increases in flavodoxin content become detrimental to plant fitness.

Stress response of transgenic tobacco plants expressing a cyanobacterial ferredoxin in chloroplasts.

Expression of the chloroplast electron shuttle ferredoxin is induced by light through mechanisms that partially depend on sequences lying in the coding region of the gene, complicating its manipulation by promoter engineering. Ferredoxin expression is also down-regulated under virtually all stress situations, and it is unclear if light-dependent induction and stress-dependent repression proceed through the same or similar mechanisms. Previous reports have shown that expression of a cyanobacterial flavodoxin in tobacco plastids results in plants with enhanced tolerance to adverse environmental conditions such as drought, chilling and xenobiotics (Tognetti et al. in Plant Cell 18:2035-2050, 2006). The protective effect of flavodoxin was linked to functional replacement of ferredoxin, suggesting the possibility that tolerant phenotypes might be obtained by simply increasing ferredoxin contents. To bypass endogenous regulatory constraints, we transformed tobacco plants with a ferredoxin gene from Anabaena sp. PCC7120, which has only 53% identity with plant orthologs. The cyanobacterial protein was able to interact in vitro with ferredoxin-dependent plant enzymes and to mediate NADP(+) photoreduction by tobacco thylakoids. Expression of Anabaena ferredoxin was constitutive and light-independent. However, homozygous lines accumulating threefold higher ferredoxin levels than the wild-type failed to show enhanced tolerance to oxidative stress and chilling temperatures. Under these adverse conditions, Anabaena ferredoxin was down-regulated even faster than the endogenous counterparts. The results indicate that: (1) light- and stress-dependent regulations of ferredoxin expression proceed through different pathways, and (2) overexpression of ferredoxin is not an alternative to flavodoxin expression for the development of increased stress tolerance in plants.

Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked-down transgenic tobacco plants.

Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin-deficient lines showed growth arrest, leaf chlorosis and decreased CO(2) assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.

An in vivo system involving co-expression of cyanobacterial flavodoxin and ferredoxin-NADP(+) reductase confers increased tolerance to oxidative stress in plants.

Oxidative stress in plants causes ferredoxin down-regulation and NADP(+) shortage, over-reduction of the photosynthetic electron transport chain, electron leakage to oxygen and generation of reactive oxygen species (ROS). Expression of cyanobacterial flavodoxin in tobacco chloroplasts compensates for ferredoxin decline and restores electron delivery to productive routes, resulting in enhanced stress tolerance. We have designed an in vivo system to optimize flavodoxin reduction and NADP(+) regeneration under stress using a version of cyanobacterial ferredoxin-NADP(+) reductase without the thylakoid-binding domain. Co-expression of the two soluble flavoproteins in the chloroplast stroma resulted in lines displaying maximal tolerance to redox-cycling oxidants, lower damage and decreased ROS accumulation. The results underscore the importance of chloroplast redox homeostasis in plants exposed to adverse conditions, and provide a tool to improve crop tolerance toward environmental hardships.