Detection and Characterization of a Novel Copper-Dependent Intermediate in a Lytic Polysaccharide Monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose which have large practical application in the process of refining biomass. The catalytic mechanism of LPMOs still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t1/2 =6-8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-CuI reacts with sub-equimolar amounts of H2 O2 . UV/Vis absorption spectroscopy, electron paramagnetic resonance, resonance Raman and stopped-flow spectroscopy suggest that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). Additionally, activity assays in the presence of sub-equimolar amounts of H2 O2 showed an increase in the LPMO oxidation of phosphoric acid swollen cellulose. Accordingly, this suggests that the long-lived copper-dependent intermediate could be part of the catalytic mechanism for LPMOs. The observed intermediate offers a new perspective into the oxidative reaction mechanism of TaLPMO9A and hence for the biomass oxidation and the reactivity of copper in biological systems.

Incidence of pediatric neuromyelitis optica spectrum disorder and myelin oligodendrocyte glycoprotein antibody-associated disease in Denmark 2008‒2018: A nationwide, population-based cohort study.

BACKGROUND: The incidence of pediatric neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease have not been reported previously. Our aim was to estimate the incidence of pediatric NMOSD and the occurrence of anti-MOG antibody-associated disease in Denmark during 2008-18, and to evaluate the diagnostic usefulness of antibodies against MOG and aquaporin-4 (AQP4) in children <18 years. METHODS: We undertook a nationwide, population-based, multicenter cohort study using data from the Danish National Patient Register, the Danish Multiple Sclerosis Registry, and laboratories providing anti-AQP4 and anti-MOG antibody analyses. Diagnoses were confirmed by review of the medical records, including blinded MRI review in most children with acute disseminated encephalomyelitis (ADEM). RESULTS: In children with acquired demyelinating syndromes, anti-AQP4 antibodies were detected in 4% and anti-MOG antibodies in 18%, including in the two children with ADEM who relapsed. We identified four children with NMOSD, equivalent to an incidence of 0.031/100,000 (95% confidence interval = 0.011‒0.082). In anti-MOG antibody-positive children, 32% relapsed during follow-up. CONCLUSIONS: Pediatric NMOSD and MOG antibody-associated disease are rare, but one-third of anti-MOG-positive children relapsed. In pediatric ADEM, only anti-MOG antibody-positive children relapsed, but the overall risk of relapse after pediatric ADEM was low.

Photosystem II Functionality in Barley Responds Dynamically to Changes in Leaf Manganese Status.

A catalytic manganese (Mn) cluster is required for the oxidation of water in the oxygen-evolving complex (OEC) of photosystem II (PSII) in plants. Despite this essential role of Mn in generating the electrons driving photosynthesis, limited information is available on how Mn deficiency affects PSII functionality. We have here used parameters derived from measurements of fluorescence induction kinetics (OJIP transients), non-photochemical quenching (NPQ) and PSII subunit composition to investigate how latent Mn deficiency changes the photochemistry in two barley genotypes differing in Mn efficiency. Mn deficiency caused dramatic reductions in the quantum yield of PSII and led to the appearance of two new inflection points, the K step and the D dip, in the OJIP fluorescence transients, indicating severe damage to the OEC. In addition, Mn deficiency decreased the ability to induce NPQ in the light, rendering the plants incapable of dissipating excess energy in a controlled way. Thus, the Mn deficient plants became severely affected in their ability to recover from high light-induced photoinhibition, especially under strong Mn deficiency. Interestingly, the Mn-efficient genotype was able to maintain a higher NPQ than the Mn-inefficient genotype when exposed to mild Mn deficiency. However, during severe Mn deficiency, there were no differences between the two genotypes, suggesting a general loss of the ability to disassemble and repair PSII. The pronounced defects of PSII activity were supported by a dramatic decrease in the abundance of the OEC protein subunits, PsbP and PsbQ in response to Mn deficiency for both genotypes. We conclude that regulation of photosynthetic performance by means of maintaining and inducing NPQ mechanisms contribute to genotypic differences in the Mn efficiency of barley genotypes growing under conditions with mild Mn deficiency.

The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1.

Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%). In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed.

Dynamic composition, shaping and organization of plastid nucleoids.

In this article recent progress on the elucidation of the dynamic composition and structure of plastid nucleoids is reviewed from a structural perspective. Plastid nucleoids are compact structures of multiple copies of different forms of ptDNA, RNA, enzymes for replication and gene expression as well as DNA binding proteins. Although early electron microscopy suggested that plastid DNA is almost free of proteins, it is now well established that the DNA in nucleoids similarly as in the nuclear chromatin is associated with basic proteins playing key roles in organization of the DNA architecture and in regulation of DNA associated enzymatic activities involved in transcription, replication, and recombination. This group of DNA binding proteins has been named plastid nucleoid associated proteins (ptNAPs). Plastid nucleoids are unique with respect to their variable number, genome copy content and dynamic distribution within different types of plastids. The mechanisms underlying the shaping and reorganization of plastid nucleoids during chloroplast development and in response to environmental conditions involve posttranslational modifications of ptNAPs, similarly to those changes known for histones in the eukaryotic chromatin, as well as changes in the repertoire of ptNAPs, as known for nucleoids of bacteria. Attachment of plastid nucleoids to membranes is proposed to be important not only for regulation of DNA availability for replication and transcription, but also for the coordination of photosynthesis and plastid gene expression.

The Arabidopsis transcription factor PHR1 is essential for adaptation to high light and retaining functional photosynthesis during phosphate starvation.

The transcription factor PHR1 (PHOSPHATE STARVATION RESPONSE 1; encoded by gene At4g28610) is central for adaptation to phosphate deficiency in Arabidopsis (Arabidopsis thaliana). A rapid turnover of phosphate pools in the leaves is essential for energy transfer and metabolism within photosynthesis, and consequently, we hypothesized that PHR1 is needed for adaptation to high-light stress during P deficiency. We analyzed three Arabidopsis plant lines: wild-type, a transgenic PHR1 overexpressor line and a knockout mutant, phr1. The plants were grown under phosphate-limiting and sufficient conditions and exposed to different light conditions. Photosynthetic activity and light stress of the leaves were characterized by analyzing accumulation of carbohydrates, chlorophyll fluorescence, immunoblot detection of photosystem subunits and anthocyanin accumulation. Compared to the wild-type and the overexpressor line, the phr1 mutant has decreased levels of phosphate, anthocyanins and carbohydrates during combined P deficiency and light stress. The stressed mutant also has strongly decreased photosystem II (PSII) quantum efficiency, and shows degradation of the core units of PSII demonstrating extensive irreversible photodamage. We conclude that PHR1 is needed for the metabolic balance, for retaining P(i) levels and for inducing anthocyanin production, and during P deficiency PHR1 is vital for adaptations to avoid permanent damage to photosystems during high-light conditions.

Manganese deficiency leads to genotype-specific changes in fluorescence induction kinetics and state transitions.

Barley (Hordeum vulgare) genotypes display a marked difference in their ability to tolerate growth at low manganese (Mn) concentrations, a phenomenon designated as differential Mn efficiency. Induction of Mn deficiency in two genotypes differing in Mn efficiency led to a decline in the quantum yield efficiency for both, although faster in the Mn-inefficient genotype. Leaf tissue and thylakoid Mn concentrations were reduced under Mn deficiency, but no difference between genotypes was observed and no visual Mn deficiency symptoms were developed. Analysis of the fluorescence induction kinetics revealed that in addition to the usual O-J-I-P steps, clear K and D steps were developed in the Mn-inefficient genotype under Mn deficiency. These marked changes indicated damages to photosystem II (PSII). This was further substantiated by state transition measurements, indicating that the ability of plants to redistribute excitation energy was reduced. The percentage change in state transitions for control plants with normal Mn supply of both genotypes was 9% to 11%. However, in Mn-deficient leaves of the Mn-inefficient genotypes, state transitions were reduced to less than 1%, whereas no change was observed for the Mn-efficient genotypes. Immunoblotting and the chlorophyll a/b ratio confirmed that Mn deficiency in general resulted in a significant reduction in abundance of PSII reaction centers relative to the peripheral antenna. In addition, PSII appeared to be significantly more affected by Mn limitation than PSI. However, the striking genotypic differences observed in Mn-deficient plants, when analyzing state transitions and fluorescence induction kinetics, could not be correlated with specific changes in photosystem proteins. Thus, there is no simple linkage between protein expression and the differential reduction in state transition and fluorescence induction kinetics observed for the genotypes under Mn deficiency.

Chlorophyll limitation in plants remodels and balances the photosynthetic apparatus by changing the accumulation of photosystems I and II through two different approaches.

Arabidopsis plants with a reduced expression of CHL27 (chl27), an enzyme (EC 1.14.13.81) required for the synthesis of Pchlide, are chlorotic and have a Chl a/b ratio two times higher than wild-type (WT). Knockdown plants transformed with a construct constitutively expressing CHL27 recovered regarding Chl level, a/b ratio and 77K fluorescence. A negative correlation was found between total Chl and Chl a/b ratio in the examined plants. The chl27 plants fail to assemble WT amounts of complete PSI and PSII, leading to an elevated PSII/PSI ratio. The PSI remaining in chl27 is fully functional with a quantum yield higher than for WT. Despite a severe reduction of photosystem II antennae protein (LHCII) and an increased proportion of stroma lammella, the chl27 plants are able to perform state transitions. No major differences were found regarding PSII quantum yield, qN and 1 - qp whereas non-photochemical quenching was decreased by a factor two in chl27 plants. The PSII quantum yield for dark-adapted plants and plants given 10 min recovery after high light treatment were similar for both WT and chl27 showing that chl27 plants are not more susceptible to photoinhibition than WT. Taken together the plant manage to acclimate and to balance the two photosystems well even when it is severely limited in Chl. The way to achieve this differs for the two photosystems: regarding PSI a general reduction of core and antenna subunits occurs with no apparent change in the antenna composition; whereas for PSII there is a preferential loss of antenna proteins.

Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum.

The plastid-encoded psaJ gene encodes a hydrophobic low-molecular-mass subunit of photosystem I (PSI) containing one transmembrane helix. Homoplastomic transformants with an inactivated psaJ gene were devoid of PSI-J protein. The mutant plants were slightly smaller and paler than wild-type because of a 13% reduction in chlorophyll content per leaf area caused by an approximately 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI-J is not involved in binding of light-harvesting complex I. The specific PSI activity, measured as NADP(+) photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second-order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI-J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis revealed a secondary loss of the luminal PSI-N subunit in PSI particles devoid of PSI-J. Presumably PSI-J affects the conformation of PSI-F, which in turn affects the binding of PSI-N. This together renders a fraction of the PSI particles inactive. Thus, PSI-J is an important subunit that, together with PSI-F and PSI-N, is required for formation of the plastocyanin-binding domain of PSI. PSI-J is furthermore important for stability or assembly of the PSI complex.

Structure of the higher plant light harvesting complex I: in vivo characterization and structural interdependence of the Lhca proteins.

We have investigated the structure of the higher plant light harvesting complex of photosystem I (LHCI) by analyzing PSI-LHCI particles isolated from a set of Arabidopsis plant lines, each lacking a specific Lhca (Lhca1-4) polypeptide. Functional antenna size measurements support the recent finding that there are four Lhca proteins per PSI in the crystal structure [Ben-Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635]. According to HPLC analyses the number of pigment molecules bound within the LHCI is higher than expected from reconstitution studies or analyses of isolated native LHCI. Comparison of the spectra of the particles from the different lines reveals chlorophyll absorption bands peaking at 696, 688, 665, and 655 nm that are not present in isolated PSI or LHCI. These bands presumably originate from "gap" or "linker" pigments that are cooperatively coordinated by the Lhca and/or PSI proteins, which we have tentatively localized in the PSI-LHCI complex.