An l-glucose Catabolic Pathway in Paracoccus Species 43P

An l-glucose-utilizing bacterium, Paracoccus sp. 43P, was isolated from soil by enrichment cultivation in a minimal medium containing l-glucose as the sole carbon source. In cell-free extracts from this bacterium, NAD⁺-dependent l-glucose dehydrogenase was detected as having sole activity toward l-glucose. This enzyme, LgdA, was purified, and the lgdA gene was found to be located in a cluster of putative inositol catabolic genes. LgdA showed similar dehydrogenase activity toward scyllo- and myo-inositols. l-Gluconate dehydrogenase activity was also detected in cell-free extracts, which represents the reaction product of LgdA activity toward l-glucose. Enzyme purification and gene cloning revealed that the corresponding gene resides in a nine-gene cluster, the lgn cluster, which may participate in aldonate incorporation and assimilation. Kinetic and reaction product analysis of each gene product in the cluster indicated that they sequentially metabolize l-gluconate to glycolytic intermediates, d-glyceraldehyde-3-phosphate, and pyruvate through reactions of C-5 epimerization by dehydrogenase/reductase, dehydration, phosphorylation, and aldolase reaction, using a pathway similar to l-galactonate catabolism in Escherichia coli. Gene disruption studies indicated that the identified genes are responsible for l-glucose catabolism.     

The initial metabolic conversion of levulinic acid in Cupriavidus necator

Levulinic acid or 4-ketovaleric acid is a potential renewable substrate for production of polyhydroxyalkanoates. In this work, the initial reactions of LA metabolism by Cupriavidus necator were examined in vitro. The organic acid was converted by membrane-bound crude enzymes obtained from the cells pre-grown on LA, while no LA activity was detected from cells pre-grown on acetic acid. Acetyl-CoA and propionyl-CoA were two major intermediates in the initial reactions of LA conversion. A mass balance on propionyl-CoA accounts for 84 mol% of LA added in vitro. It explains an interesting phenomenon that 3-hydroxbutyrate and 3-hydroxyvalerate are two major monomers of the biopolyester formed from LA, instead of 4-hydroxvalerate that has the similar chemical structure of LA as the precursor. A Monod model was used to describe the kinetics of LA utilization as a sole carbon source or a co-substrate of glucose and fructose. The μ_max and K_m of LA alone were 0.26 h⁻¹ and 0.01 g/L, respectively. The content and composition of PHA are also dependent on the culture conditions such as carbon to nitrogen ratio. The in vitro observation is supported by the high utilization rate of LA and the high molar percentage of 3HB and 3HV in the PHA derived from LA.     

Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis: IDENTIFICATION AND CHARACTERIZATION OF THE GE81112 BIOSYNTHETIC GENE CLUSTER*

The GE81112 tetrapeptides (1–3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.     

Exometabolome analysis identifies pyruvate dehydrogenase as a target for the antibiotic triphenylbismuthdichloride in multiresistant bacterial pathogens

The desperate need for new therapeutics against notoriously antibiotic-resistant bacteria has led to a quest for novel antibacterial target structures and compounds. Moreover, defining targets and modes of action of new antimicrobial compounds remains a major challenge with standard technologies. Here we characterize the antibacterial properties of triphenylbismuthdichloride (TPBC), which has recently been successfully used against device-associated infections. We demonstrate that TPBC has potent antimicrobial activity against many bacterial pathogens. Using an exometabolome profiling approach, a unique TPBC-mediated change in the metabolites of Staphylococcus aureus was identified, indicating that TPBC blocks bacterial pyruvate catabolism. Enzymatic studies showed that TPBC is a highly efficient, uncompetitive inhibitor of the bacterial pyruvate dehydrogenase complex. Our study demonstrates that metabolomics approaches can offer new avenues for studying the modes of action of antimicrobial compounds, and it indicates that inhibition of the bacterial pyruvate dehydrogenase complex may represent a promising strategy for combating multidrug-resistant bacteria.     

Genetic and biochemical characterization of a pathway for the degradation of 2-aminoethylphosphonate in Sinorhizobium meliloti 1021

A variety of microorganisms have the ability to use phosphonic acids as sole sources of phosphorus. Here, a novel pathway for degradation of 2-aminoethylphosphonate in the bacterium Sinorhizobium meliloti 1021 is proposed based on the analysis of the genome sequence. Gene deletion experiments confirmed the involvement of the locus containing phnW, phnA, and phnY genes in the conversion of 2-aminoethylphosphonate to inorganic phosphate. Biochemical studies of the recombinant PhnY and PhnA proteins verified their roles as phosphonoacetaldehyde dehydrogenase and phosphonoacetate hydrolase, respectively. This pathway is likely not limited to S. meliloti as suggested by the presence of homologous gene clusters in other bacterial genomes.     

Identification and characterization of oxalate oxidoreductase, a novel thiamine pyrophosphate-dependent 2-oxoacid oxidoreductase that enables anaerobic growth on oxalate

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO(2) or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe(4)S(4)] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a k(cat) of 0.09 s(-1) and a K(m) of 58 μM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood-Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism.     

Role of serine/threonine phosphatase (SP-STP) in Streptococcus pyogenes physiology and virulence

Reversible phosphorylation is the key mechanism regulating several cellular events in prokaryotes and eukaryotes. In prokaryotes, signal transduction is perceived to occur primarily via the two-component signaling system involving histidine kinases and cognate response regulators. Although an alternative regulatory pathway controlled by the eukaryote-type serine/threonine kinase (Streptococcus pyogenes serine/threonine kinase; SP-STK) has been shown to modulate bacterial growth, division, adherence, invasion, and virulence in group A Streptococcus (GAS; S. pyogenes), the precise role of the co-transcribing serine/threonine phosphatase (SP-STP) has remained enigmatic. In this context, this is the first report describing the construction and characterization of non-polar SP-STP mutants in two different strains of Type M1 GAS. The STP knock-out mutants displayed increased bacterial chain lengths in conjunction with thickened cell walls, significantly reduced capsule and hemolysin production, and restoration of the phenotypes postcomplementation. The present study also reveals important contribution of cognately regulated-reversible phosphorylation by SP-STK/SP-STP on two major response regulators of two-component systems, WalRK and CovRS. We also demonstrate a distinct role of SP-STP in terms of expression of surface proteins and SpeB in a strain-specific manner. Further, the attenuation of virulence in the absence of STP and its restoration only in the complemented strains that were generated by the use of a low copy plasmid and not by a high copy one emphasize not only the essential role of STP in virulence but also highlight the tightly regulated SP-STP/SP-STK-mediated cognate functions. SP-STP thus is an important regulator of GAS virulence and plays a critical role in GAS pathogenesis.     

Arrested oocyst maturation in Plasmodium parasites lacking type II NADH:ubiquinone dehydrogenase

The Plasmodium mitochondrial electron transport chain has received considerable attention as a potential target for new antimalarial drugs. Atovaquone, a potent inhibitor of Plasmodium cytochrome bc(1), in combination with proguanil is recommended for chemoprophylaxis and treatment of malaria. The type II NADH:ubiquinone oxidoreductase (NDH2) is considered an attractive drug target, as its inhibition is thought to lead to the arrest of the mitochondrial electron transport chain and, as a consequence, pyrimidine biosynthesis, an essential pathway for the parasite. Using the rodent malaria parasite Plasmodium berghei as an in vivo infection model, we studied the role of NDH2 during Plasmodium life cycle progression. NDH2 can be deleted by targeted gene disruption and, thus, is dispensable for the pathogenic asexual blood stages, disproving the candidacy for an anti-malarial drug target. After transmission to the insect vector, NDH2-deficient ookinetes display an intact mitochondrial membrane potential. However, ndh2(-) parasites fail to develop into mature oocysts in the mosquito midgut. We propose that Plasmodium blood stage parasites rely on glycolysis as the main ATP generating process, whereas in the invertebrate vector, a glucose-deprived environment, the malaria parasite is dependent on an intact mitochondrial respiratory chain.     

NAT8L (N-Acetyltransferase 8-Like) Accelerates Lipid Turnover and Increases Energy Expenditure in Brown Adipocytes

NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes.     

The Urokinase Receptor Homolog Haldisin Is a Novel Differentiation Marker of Stratum Granulosum in Squamous Epithelia

Several members of the Ly-6/uPAR (LU)-protein domain family are differentially expressed in human squamous epithelia. In some cases, they even play important roles in maintaining skin homeostasis, as exemplified by the secreted single domain member, SLURP-1, the deficiency of which is associated with the development of palmoplantar hyperkeratosis in the congenital skin disorder Mal de Meleda. In the present study, we have characterized a new member of the LU-protein domain family, which we find to be predominantly expressed in the stratum granulosum of human skin, thus resembling the expression of SLURP-1. In accordance with its expression pattern, we denote this protein product, which is encoded by the LYPD5 gene, as Haldisin (human antigen with LU-domains expressed in skin). Two of the five human glycolipid-anchored membrane proteins with multiple LU-domains characterized so far are predominantly confined to squamous epithelia (i.e., C4.4A), to stratum spinosum, and Haldisin to stratum granulosum under normal homeostatic conditions. Whether Haldisin is a prognostic biomarker for certain epithelial malignancies, like C4.4A and SLURP-1, remains to be explored.     

Copper Import into the Mitochondrial Matrix in Saccharomyces cerevisiae Is Mediated by Pic2, a Mitochondrial Carrier Family Protein

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.     

ubiI,a New Gene in Escherichia coli Coenzyme Q Biosynthesis,Is Involved in Aerobic C5-hydroxylation

Coenzyme Q (ubiquinone or Q) is a redox-active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy-generating processes. Q biosynthesis is a complex pathway that involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli, and we have renamed it ubiI. Based on genetic and biochemical experiments, we establish that the UbiI protein functions in the C5-hydroxylation reaction. A strain deficient in ubiI has a low level of Q and accumulates a compound derived from the Q biosynthetic pathway, which we purified and characterized. We also demonstrate that UbiI is only implicated in aerobic Q biosynthesis and that an alternative enzyme catalyzes the C5-hydroxylation reaction in the absence of oxygen. We have solved the crystal structure of a truncated form of UbiI. This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. Site-directed mutagenesis confirms that residues of the flavin binding pocket of UbiI are important for activity. With our identification of UbiI, the three monooxygenases necessary for aerobic Q biosynthesis in E. coli are known.     

Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases

Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Δhgprt/Δxprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084–16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the Δhgprt/Δxprt knock-out cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2′-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of Δhgprt/Δxprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which Δhgprt/Δxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Δhgprt/Δxprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of Δhgprt/Δxprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Δhgprt/Δxprt parasites displayed persistence and increased virulence in mice.     

Multidrug-resistant phenotype and isolation of a Novel SHV- beta-Lactamase variant in a clinical isolate of Enterobacter cloacae

Background

ESBL-producing bacteria are a clinical problem in the management of diseases caused by these pathogens. Worldwide, systemic infections with BL enzymes are evolving by mutations from classical bla genes in an intensified manner and they continue to be transferred across species.

Results

E.cloacae BF1417 isolate and its transconjugants gave positive results with the DDST, suggesting the presence of ESBL. Sequence analysis revealed a bla_SHV-ESBL-type gene that differs from the gene encoding SHV-1 by five point mutations resulting in three amino acid substitutions in the coding region: C123R, I282T and L286P. This novel SHV-type enzyme was designated SHV-128. The conjugation tests and plasmid characterization showed that the bla_SHV-128 is located on a conjugative plasmid IncFII type. Expression studies demonstrated that the above mutations participated in drug resistance, hydrolysis of extended spectrum β-lactam and the change of the isoelectric point of the protein.

Conclusion

These findings underscore the diversity by which antibiotic resistance can arise and the evolutionary potential of the clinically important ESBL enzymes. In addition, this study highlights the need for systematic surveillance of ESBL-mediated resistance as well as in clinical areas and communities.     

A sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli

l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity.     

H3K27 Trimethylation Is an Early Epigenetic Event of p16INK4a Silencing for Regaining Tumorigenesis in Fusion Reprogrammed Hepatoma Cells

Stable epigenetic silencing of p16^INK4a is a common event in hepatocellular carcinoma (HCC) cells, which is associated with abnormal cell proliferation and liberation from cell cycle arrest. Understanding the early epigenetic events in silencing p16^INK4a expression may illuminate a prognostic strategy to block HCC development. Toward this end, we created a reprogram cell model by the fusion mouse HCC cells with mouse embryonic stem cells, in which the ES-Hepa hybrids forfeited HCC cell characteristics along with reactivation of the silenced p16^INK4a. HCC characteristics, in terms of gene expression pattern and tumorigenic potential, was restored upon induced differentiation of these reprogrammed ES-Hepa hybrids. The histone methylation pattern relative to p16^INK4a silencing during differentiation of the ES-Hepa hybrids was analyzed. H3K27 trimethylation at the p16^INK4a promoter region, occurring in the early onset of p16^INK4a silencing, was followed by H3K9 dimethylation at later stages. During the induced differentiation of the ES-Hepa hybrids, H3K4 di- and trimethylations were maintained at high levels during the silencing of p16^INK4a, strongly suggesting that H3K4 methylation events did not cause the silencing of p16^INK4a. Our results suggested that the enrichment of H3K27 trimethylation, independent of H3K9 dimethylation, trimethylation, and DNA methylation, was an early event in the silencing of p16^INK4a during the tumor development. This unique chromatin pattern may be a heritable marker of epigenetic regulation for p16^INK4a silencing during the developmental process of hepatocellular carcinogenesis.     

Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum

The photosynthetic green sulfur bacterium Chlorobaculum tepidum assimilates CO(2) and organic carbon sources (acetate or pyruvate) during mixotrophic growth conditions through a unique carbon and energy metabolism. Using a (13)C-labeling approach, this study examined biosynthetic pathways and flux distributions in the central metabolism of C. tepidum. The isotopomer patterns of proteinogenic amino acids revealed an alternate pathway for isoleucine synthesis (via citramalate synthase, CimA, CT0612). A (13)C-assisted flux analysis indicated that carbons in biomass were mostly derived from CO(2) fixation via three key routes: the reductive tricarboxylic acid (RTCA) cycle, the pyruvate synthesis pathway via pyruvate:ferredoxin oxidoreductase, and the CO(2)-anaplerotic pathway via phosphoenolpyruvate carboxylase. During mixotrophic growth with acetate or pyruvate as carbon sources, acetyl-CoA was mainly produced from acetate (via acetyl-CoA synthetase) or citrate (via ATP citrate lyase). Pyruvate:ferredoxin oxidoreductase converted acetyl-CoA and CO(2) to pyruvate, and this growth-rate control reaction is driven by reduced ferredoxin generated during phototrophic growth. Most reactions in the RTCA cycle were reversible. The relative fluxes through the RTCA cycle were 80～100 units for mixotrophic cultures grown on acetate and 200～230 units for cultures grown on pyruvate. Under the same light conditions, the flux results suggested a trade-off between energy-demanding CO(2) fixation and biomass growth rate; C. tepidum fixed more CO(2) and had a higher biomass yield (Y(X/S), mole carbon in biomass/mole substrate) in pyruvate culture (Y(X/S) = 9.2) than in acetate culture (Y(X/S) = 6.4), but the biomass growth rate was slower in pyruvate culture than in acetate culture.     

Crystal structure of HugZ, a novel heme oxygenase from Helicobacter pylori

The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His²⁴⁵ and an azide molecule when it is present in crystallization conditions. Experiments show that Arg¹⁶⁶, which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His²⁴⁵, surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.     

The Photocycle and Proton Translocation Pathway in a Cyanobacterial Ion-Pumping Rhodopsin

The genome of thylakoidless cyanobacterium Gloeobacter violaceus encodes a fast-cycling rhodopsin capable of light-driven proton transport. We characterize the dark state, the photocycle, and the proton translocation pathway of GR spectroscopically. The dark state of GR contains predominantly all-trans-retinal and, similar to proteorhodopsin, does not show the light/dark adaptation. We found an unusually strong coupling between the conformation of the retinal and the site of Glu¹³², the homolog of Asp⁹⁶ of BR. Although the photocycle of GR is similar to that of proteorhodopsin in general, it differs in accumulating two intermediates typical for BR, the L-like and the N-like states. The latter state has a deprotonated cytoplasmic proton donor and is spectrally distinct from the strongly red-shifted N intermediate known for proteorhodopsin. The proton uptake precedes the release and occurs during the transition to the O intermediate. The proton translocation pathway of GR is similar to those of other proton-pumping rhodopsins, involving homologs of BR Schiff base proton acceptor and donor Asp⁸⁵ and Asp⁹⁶ (Asp¹²¹ and Glu¹³²). We assigned a pair of FTIR bands (positive at 1749 cm⁻¹ and negative at 1734 cm⁻¹) to the protonation and deprotonation, respectively, of these carboxylic acids.