Spectral modulation of B850 bacteriochlorophyll a in light-harvesting complex 2 from purple photosynthetic bacterium Thermochromatium tepidum by detergents and calcium ions.

Spectral variations of light-harvesting (LH) proteins of purple photosynthetic bacteria provide insight into the molecular mechanisms underlying spectral tuning of circular bacteriochlorophyll (BChl) arrays, which play crucial roles in photoenergy conversion in these organisms. Here we investigate spectral changes of the Qy band of B850 BChl a in LH2 protein from purple sulfur bacterium Thermochromatium tepidum (tepidum-LH2) by detergents and Ca2+. The tepidum-LH2 solubilized with lauryl dimethylamine N-oxide and n-octyl-ß-D-glucoside (LH2LDAO and LH2OG, respectively) exhibited blue-shift of the B850 Qy band with hypochromism compared with the tepidum-LH2 solubilized with n-dodecyl-ß-D-maltoside (LH2DDM), resulting in the LH3-like spectral features. Resonance Raman spectroscopy indicated that this blue-shift was ascribable to the loss of hydrogen-bonding between the C3-acetyl group in B850 BChl a and the LH2 polypeptides. Ca2+ produced red-shift of the B850 Qy band in LH2LDAO by forming hydrogen-bond for the C3-acetyl group in B850 BChl a, probably due to a change in the microenvironmental structure around B850. Ca2+-induced red-shift was also observed in LH2OG although the B850 acetyl group is still free from hydrogen-bonding. Therefore, the Ca2+-induced B850 red-shift in LH2OG would originate from an electrostatic effect of Ca2+. The current results suggest that the B850 Qy band in tepidum-LH2 is primarily tuned by two mechanisms, namely the hydrogen-bonding of the B850 acetyl group and the electrostatic effect.

High-resolution structure and biochemical properties of the LH1-RC photocomplex from the model purple sulfur bacterium, Allochromatium vinosum.

The mesophilic purple sulfur phototrophic bacterium Allochromatium (Alc.) vinosum (bacterial family Chromatiaceae) has been a favored model for studies of bacterial photosynthesis and sulfur metabolism, and its core light-harvesting (LH1) complex has been a focus of numerous studies of photosynthetic light reactions. However, despite intense efforts, no high-resolution structure and thorough biochemical analysis of the Alc. vinosum LH1 complex have been reported. Here we present cryo-EM structures of the Alc. vinosum LH1 complex associated with reaction center (RC) at 2.24 Å resolution. The overall structure of the Alc. vinosum LH1 resembles that of its moderately thermophilic relative Alc. tepidum in that it contains multiple pigment-binding α- and ß-polypeptides. Unexpectedly, however, six Ca ions were identified in the Alc. vinosum LH1 bound to certain α1/ß1- or α1/ß3-polypeptides through a different Ca2+-binding motif from that seen in Alc. tepidum and other Chromatiaceae that contain Ca2+-bound LH1 complexes. Two water molecules were identified as additional Ca2+-coordinating ligands. Based on these results, we reexamined biochemical and spectroscopic properties of the Alc. vinosum LH1-RC. While modest but distinct effects of Ca2+ were detected in the absorption spectrum of the Alc. vinosum LH1 complex, a marked decrease in thermostability of its LH1-RC complex was observed upon removal of Ca2+. The presence of Ca2+ in the photocomplex of Alc. vinosum suggests that Ca2+-binding to LH1 complexes may be a common adaptation in species of Chromatiaceae for conferring spectral and thermal flexibility on this key component of their photosynthetic machinery.

Structural insights into the unusual core photocomplex from a triply extremophilic purple bacterium, Halorhodospira halochloris.

Halorhodospira (Hlr.) halochloris is a triply extremophilic phototrophic purple sulfur bacterium, as it is thermophilic, alkaliphilic, and extremely halophilic. The light-harvesting-reaction center (LH1-RC) core complex of this bacterium displays an LH1-Qy transition at 1,016 nm, which is the lowest-energy wavelength absorption among all known phototrophs. Here we report the cryo-EM structure of the LH1-RC at 2.42 Å resolution. The LH1 complex forms a tricyclic ring structure composed of 16 αßγ-polypeptides and one αß-heterodimer around the RC. From the cryo-EM density map, two previously unrecognized integral membrane proteins, referred to as protein G and protein Q, were identified. Both of these proteins are single transmembrane-spanning helices located between the LH1 ring and the RC L-subunit and are absent from the LH1-RC complexes of all other purple bacteria of which the structures have been determined so far. Besides bacteriochlorophyll b molecules (B1020) located on the periplasmic side of the Hlr. halochloris membrane, there are also two arrays of bacteriochlorophyll b molecules (B800 and B820) located on the cytoplasmic side. Only a single copy of a carotenoid (lycopene) was resolved in the Hlr. halochloris LH1-α3ß3 and this was positioned within the complex. The potential quinone channel should be the space between the LH1-α3ß3 that accommodates the single lycopene but does not contain a γ-polypeptide, B800 and B820. Our results provide a structural explanation for the unusual Qy red shift and carotenoid absorption in the Hlr. halochloris spectrum and reveal new insights into photosynthetic mechanisms employed by a species that thrives under the harshest conditions of any phototrophic microorganism known.

Calcium and the ecology of photosynthesis in purple sulfur bacteria.

The ecological success of purple sulfur bacteria (PSB) is linked to their ability to collect near-infrared solar energy by membrane-integrated, pigment-protein photocomplexes. These include a Core complex containing both light-harvesting 1 (LH1) and reaction centre (RC) components (called the LH1-RC photocomplex) present in all PSB and a peripheral light-harvesting complex present in most but not all PSB. In research to explain the unusual absorption properties of the thermophilic purple sulfur bacterium Thermochromatium tepidum, Ca2+ was discovered bound to LH1 polypeptides in its LH1-RC; further work showed that calcium controls both the thermostability and unusual spectrum of the Core complex. Since then, Ca2+ has been found in the LH1-RC photocomplexes of several other PSB, including mesophilic species, but not in the LH1-RC of purple non-sulfur bacteria. Here we focus on four species of PSB-two thermophilic and two mesophilic-and describe how Ca2+ is integrated into and affects their photosynthetic machinery and why this previously overlooked divalent metal is a key nutrient for their ecological success.

Heterologous expression and characterization of salt-tolerant ß-glucosidase from xerophilic Aspergillus chevalieri for hydrolysis of marine biomass.

A salt-tolerant exo-ß-1,3-glucosidase (BGL_MK86) was cloned from the xerophilic mold Aspergillus chevalieri MK86 and heterologously expressed in A. oryzae. Phylogenetic analysis suggests that BGL_MK86 belongs to glycoside hydrolase family 5 (aryl-phospho-ß-D-glucosidase, BglC), and exhibits D-glucose tolerance. Recombinant BGL_MK86 (rBGL_MK86) exhibited 100-fold higher expression than native BGL_MK86. rBGL_MK86 was active over a wide range of NaCl concentrations [0%-18% (w/v)] and showed increased substrate affinity for p-nitrophenyl-ß-D-glucopyranoside (pNPBG) and turnover number (kcat) in the presence of NaCl. The enzyme was stable over a broad pH range (5.5-9.5). The optimum reaction pH and temperature for hydrolysis of pNPBG were 5.5 and 45 °C, respectively. rBGL_MK86 acted on the ß-1,3-linked glucose dimer laminaribiose, but not ß-1,4-linked or ß-1,6-linked glucose dimers (cellobiose or gentiobiose). It showed tenfold higher activity toward laminarin (a linear polymer of ß-1,3 glucan) from Laminaria digitata than laminarin (ß-1,3/ß-1,6 glucan) from Eisenia bicyclis, likely due to its inability to act on ß-1,6-linked glucose residues. The ß-glucosidase retained hydrolytic activity toward crude laminarin preparations from marine biomass in moderately high salt concentrations. These properties indicate wide potential applications of this enzyme in saccharification of salt-bearing marine biomass.

Genomic basis for the unique phenotype of the alkaliphilic purple nonsulfur bacterium Rhodobaca bogoriensis.

Although several species of purple sulfur bacteria inhabit soda lakes, Rhodobaca bogoriensis is the first purple nonsulfur bacterium cultured from such highly alkaline environments. Rhodobaca bogoriensis strain LBB1T was isolated from Lake Bogoria, a soda lake in the African Rift Valley. The phenotype of Rhodobaca bogoriensis is unique among purple bacteria; the organism is alkaliphilic but not halophilic, produces carotenoids absent from other purple nonsulfur bacteria, and is unable to grow autotrophically or fix molecular nitrogen. Here we analyze the draft genome sequence of Rhodobaca bogoriensis to gain further insight into the biology of this extremophilic purple bacterium. The strain LBB1T genome consists of 3.91 Mbp with no plasmids. The genome sequence supports the defining characteristics of strain LBB1T, including its (1) production of a light-harvesting 1-reaction center (LH1-RC) complex but lack of a peripheral (LH2) complex, (2) ability to synthesize unusual carotenoids, (3) capacity for both phototrophic (anoxic/light) and chemotrophic (oxic/dark) energy metabolisms, (4) utilization of a wide variety of organic compounds (including acetate in the absence of a glyoxylate cycle), (5) ability to oxidize both sulfide and thiosulfate despite lacking the capacity for autotrophic growth, and (6) absence of a functional nitrogen-fixation system for diazotrophic growth. The assortment of properties in Rhodobaca bogoriensis has no precedent among phototrophic purple bacteria, and the results are discussed in relation to the organism's soda lake habitat and evolutionary history.

Improvement in salt-tolerance of Aspergillus oryzae γ-glutamyl transpeptidase via protein chimerization with Aspergillus sydowii homolog.

γ-Glutamyl transpeptidase is one of the key enzymes involved in glutamate production during high-salt fermentation of soy sauce and miso by koji mold, Aspergillus oryzae. However, the activity of γ-glutamyl transpeptidase from A. oryzae (AOggtA) is markedly reduced in the presence of NaCl, thus classifying it as a non-salt-tolerant enzyme. In contrast, the homologous protein from the xerophilic mold, A. sydowii (ASggtA) maintains its activity under high-salt conditions. Therefore, in this study, a chimeric enzyme, ASAOggtA, was designed and engineered to improve salt-tolerance in AOggtA by swapping the N-terminal region, based on sequence and structure comparisons between salt-tolerant ASggtA and non-salt-tolerant AOggtA. The parental AOggtA and ASggtA and their chimera, ASAOggtA, were heterologously expressed in A. oryzae and purified. The chimeric enzyme inherited the superior activity and stability from each of the two parent enzymes. ASAOggtA showed > 2-fold greater tolerance than AOggtA in the presence of 18% NaCl. In addition, the chimera showed a broader range of pH stability and greater thermostability than ASggtA. AOggtA and ASAOggtA were sy over the range pH 3.0 to pH 10.5. Thermal stability was found to be in the order AOggtA (57.5 °C, t1/2 = 32.5 min) > ASAOggtA (55 °C, t1/2 = 20.5 min) > ASggtA (50 °C, t1/2 = 12.5 min). The catalytic and structural characteristics indicated that non-salt-tolerant AOggtA would not undergo irreversible structural changes in the presence of NaCl, but rather a temporary conformational change, which might result in reducing the substrate binding and catalytic activity, on the basis of kinetic properties. In addition, the chimeric enzyme showed hydrolytic activity toward L-glutamine that was as high as that of AOggtA. The newly-designed chimeric ASAOggtA might have potential applications in high-salt fermentation, such as miso and shoyu, to increase the content of the umami-flavor amino acid, L-glutamate.

Atomic force microscopic analysis of the light-harvesting complex 2 from purple photosynthetic bacterium Thermochromatium tepidum.

Structural information on the circular arrangements of repeating pigment-polypeptide subunits in antenna proteins of purple photosynthetic bacteria is a clue to a better understanding of molecular mechanisms for the ring-structure formation and efficient light harvesting of such antennas. Here, we have analyzed the ring structure of light-harvesting complex 2 (LH2) from the thermophilic purple bacterium Thermochromatium tepidum (tepidum-LH2) by atomic force microscopy. The circular arrangement of the tepidum-LH2 subunits was successfully visualized in a lipid bilayer. The average top-to-top distance of the ring structure, which is correlated with the ring size, was 4.8 ± 0.3 nm. This value was close to the top-to-top distance of the octameric LH2 from Phaeospirillum molischianum (molischianum-LH2) by the previous analysis. Gaussian distribution of the angles of the segments consisting of neighboring subunits in the ring structures of tepidum-LH2 yielded a median of 44°, which corresponds to the angle for the octameric circular arrangement (45°). These results indicate that tepidum-LH2 has a ring structure consisting of eight repeating subunits. The coincidence of an octameric ring structure of tepidum-LH2 with that of molischianum-LH2 is consistent with the homology of amino acid sequences of the polypeptides between tepidum-LH2 and molischianum-LH2.

Rhodobacter capsulatus forms a compact crescent-shaped LH1-RC photocomplex.

Rhodobacter (Rba.) capsulatus has been a favored model for studies of all aspects of bacterial photosynthesis. This purple phototroph contains PufX, a polypeptide crucial for dimerization of the light-harvesting 1-reaction center (LH1-RC) complex, but lacks protein-U, a U-shaped polypeptide in the LH1-RC of its close relative Rba. sphaeroides. Here we present a cryo-EM structure of the Rba. capsulatus LH1-RC purified by DEAE chromatography. The crescent-shaped LH1-RC exhibits a compact structure containing only 10 LH1 αß-subunits. Four αß-subunits corresponding to those adjacent to protein-U in Rba. sphaeroides were absent. PufX in Rba. capsulatus exhibits a unique conformation in its N-terminus that self-associates with amino acids in its own transmembrane domain and interacts with nearby polypeptides, preventing it from interacting with proteins in other complexes and forming dimeric structures. These features are discussed in relation to the minimal requirements for the formation of LH1-RC monomers and dimers, the spectroscopic behavior of both the LH1 and RC, and the bioenergetics of energy transfer from LH1 to the RC.

Advances in the Spectroscopic and Structural Characterization of Core Light-Harvesting Complexes from Purple Phototrophic Bacteria.

Purple phototrophic bacteria are ancient anoxygenic phototrophs and attractive research tools because they capture light energy in the near-infrared (NIR) region of the spectrum and transform it into chemical energy by way of uphill energy transfers. The heart of this reaction occurs in light-harvesting 1-reaction center (LH1-RC) complexes, which are the simplest model systems for understanding basic photosynthetic reactions within type-II (quinone-utilizing) reaction centers. In this Perspective, we highlight structure-function relationships concerning unresolved fundamental processes in purple bacterial photosynthesis, including the diversified light-harvesting capacity of LH1-associated BChl molecules, energies necessary for photoelectric conversion in the RC special pairs, and quinone transport mechanisms. Based on recent progress in the spectroscopic and structural analysis of LH1-RC complexes from a variety of purple phototrophs, we discuss several key factors for understanding how purple bacteria resource light energy in the inherently energy-poor NIR region of the electromagnetic spectrum.