Identification of a lycopene beta-cyclase required for bacteriorhodopsin biogenesis in the archaeon Halobacterium salinarum

Biogenesis of the light-driven proton pump bacteriorhodopsin in the archaeon Halobacterium salinarum requires coordinate synthesis of the bacterioopsin apoprotein and carotenoid precursors of retinal, which serves as a covalently bound cofactor. As a step towards elucidating the mechanism and regulation of carotenoid metabolism during bacteriorhodopsin biogenesis, we have identified an H. salinarum gene required for conversion of lycopene to beta-carotene, a retinal precursor. The gene, designated crtY, is predicted to encode an integral membrane protein homologous to lycopene beta-cyclases identified in bacteria and fungi. To test crtY function, we constructed H. salinarum strains with in-frame deletions in the gene. In the deletion strains, bacteriorhodopsin, retinal, and beta-carotene were undetectable, whereas lycopene accumulated to high levels ( approximately 1.3 nmol/mg of total cell protein). Heterologous expression of H. salinarum crtY in a lycopene-producing Escherichia coli strain resulted in beta-carotene production. These results indicate that H. salinarum crtY encodes a functional lycopene beta-cyclase required for bacteriorhodopsin biogenesis. Comparative sequence analysis yields a topological model of the protein and provides a plausible evolutionary connection between heterodimeric lycopene cyclases in bacteria and bifunctional lycopene cyclase-phytoene synthases in fungi.     

Different proteins bind to the butyrolactone receptor protein ARE sequence located upstream of the regulatory ccaR gene of Streptomyces clavuligerus

Cell-free extracts from Streptomyces clavuligerus, purified by elution from heparin-agarose with an ARE-containing DNA fragment or by salt elution chromatography, bind to a 26 nt ARE sequence, for butyrolactone receptor proteins (ARE(ccaR)). This sequence is [corrected] located upstream of the ccaR gene, encoding [corrected] the activator protein CcaR required for clavulanic acid and cephamycin C biosynthesis. The binding is specific for the ARE sequence as shown by competition with a 34 nt unlabelled probe identical to the ARE sequence. A brp gene, encoding a butyrolactone receptor protein, was cloned from S. clavuligerus. Sixty-one nucleotides upstream of brp another ARE sequence (ARE(brp)) was found, suggesting that Brp autoregulates its expression. Pure recombinant rBrp protein binds specifically to the ARE sequences present upstream of ccaR and brp. A brp-deleted mutant, S. clavuligerus Deltabrp::neo1, produced 150-300% clavulanic acid and 120-220% cephamycin C as compared with the parental strain, suggesting that Brp exerts a repressor role in antibiotic biosynthesis. EMSA assays using affinity chromatography extracts from the deletion mutant S. clavuligerus Deltabrp::neo1 lacked a high-mobility band-shift due to Brp but still showed a [corrected] slow-mobility band-shift observed in the wild-type strain. These results indicate that two different proteins bind specifically to the ARE sequence and modulate clavulanic acid and cephamycin C [corrected] biosynthesis by its action on ccaR gene expression.     

Bioorganic chemistry of the purple membrane ofHalobacterium halobium — Chromophore and apoprotein modified bacteriorhodopsins

Iodophenyl and anthryl retinal analogues have been synthesized. Thetrans-isomers have been isolated and purified by high pressure liquid chromatography. The purified isomers have been further characterized by nuclear magnetic resonance and ultraviolet-visible spectroscopy. Incubation of these retinal analogues with apoprotein (bacterioopsin), isolated from the purple membrane ofHalobacterium halobium gave new bacteriorhodopsin analogues. These analogues have been investigated for their absorption properties and stability. The iodophenyl analogue has been found to bind to bacterioopsin rapidly. The pigment obtained from this analogue showed a dramatically altered opsin shift of 1343 cm^-1. The anthryl analogue based bacteriorhodopsin, however, showed an opsin shift of 3849 cm^-1. It has been found that bacteriorhodopsin is quite unrestrictive in the ionone ring site. The apoprotein seems to prefer chromophores that have the ring portion co-planar with the polyene side chain. The purple membrane has also been modified by treatment with fluorescamine, a surface active reagent specific for amino groups. Reaction under controlled stoichiometric conditions resulted in the formation of a modified pigment. The new pigment showed a band at 390 nm—indicative of fluorescamine reaction with amino group (s) of apoprotein-besides retaining its original absorption band at 560 nm. Analysis of the fluorescamine modified bacteriorhodopsin resulted in the identification of lysine 129 as the modified amino acid residue. Fluorescamine-modified-bacteriorhodopsin suspension did not release protons under photolytic conditions. However, proteoliposomes of fluorescamine-modified-bacteriorhodopsin were found to show proton uptake, though at a reduced rate. Presented at the 3rd National Symposium on Bioorganic Chemistry, 1987, Hyderabad.     

Effects of light and low oxygen tension on pigment biosynthesis in Halobacterium salinarum, revealed by a novel method to quantify both retinal and carotenoids

A novel method for analyzing halobacterial pigments was developed, in which retinal was liberated from halobacterial rhodopsins as retinal oxime by hydroxylamine, ethyl beta-apo-8'-carotenoate was introduced as an internal standard, and the pigments including bacterioruberin and beta-carotene were analyzed by HPLC at the same time. With this method, we revealed that light enhances the biosynthesis of bacterioruberin and the conversion of beta-carotene to retinal, but does not affect beta-carotene biosynthesis in Halobacterium salinarum strain Oyon Moussa-16. Low oxygen tension given in the light brought a slight increase in retinal accumulation, although its biosynthesis from beta-carotene is an oxygenation reaction. This paradox could be explained by the increase in beta-carotene biosynthesis.     

Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all-trans retinal. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly alpha-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.     

Bacterioopsin, haloopsin, and sensory opsin I of the halobacterial isolate Halobacterium sp. strain SG1: three new members of a growing family.

The genes coding for bacterioopsin, haloopsin, and sensory opsin I of a halobacterial isolate from the Red Sea called Halobacterium sp. strain SG1 have been cloned and sequenced. The deduced protein sequences were aligned to the previously known halobacterial retinal proteins. The addition of these new sequences lowered the number of conserved residues to only 23 amino acids, or 8% of the alignment. Data base searches with two highly conserved peptides as well as with an alignment profile yielded no significant similarity to any other protein, so the halobacterial retinal proteins should be regarded as a distinct protein family. The protein alignment was used to make predictions about the structure of the retinal proteins as well as about the amino acids in contact with retinal proteins. These results were in excellent agreement with the structural model of bacteriorhodopsin of Halobacterium halobium as well as with mutant studies, indicating that (i) structure predictions based on the sequences of a membrane protein family can be quite accurate; (ii) halorhodopsin and sensory rhodopsin I have tertiary structures similar to that of bacteriorhodopsin; (iii) conserved amino acids do not take part in reactions specific for one group of proteins, e.g., proton translocation for bacteriorhodopsins, but have a crucial role in determining the conformation and reactions of the chromophore; and (iv) the general mode of action (light-induced chromophore and protein movements) is the same for all halobacterial retinal proteins, ion pumps as well as sensors.     

Halobacterium sp. GRB: a species to work with!?

The properties of the halobacterial isolate Halobacterium sp. GRB are discussed, especially in relation to its use as a laboratory strain. Experimental results on this species are described, including the isolation of point mutants in the bacterioopsin gene leading to single amino acid replacements in bacteriorhodopsin, the application of a selection procedure for the isolation of different types of mutants, the genetic stability of Halobacterium sp. GRB and the possibility of isolating a set of isogenic mutants, the conditions for transformation experiments with this species, and specific features of Halobacterium sp. GRB, such as halocin production and the absence of a restriction system, as well as DNA adenosine methylation.     

Bacterioopsin-mediated regulation of bacterioruberin biosynthesis in Halobacterium salinarum

Integral membrane protein complexes consisting of proteins and small molecules that act as cofactors have important functions in all organisms. To form functional complexes, cofactor biosynthesis must be coordinated with the production of corresponding apoproteins. To examine this coordination, we study bacteriorhodopsin (BR), a light-induced proton pump in the halophilic archaeon Halobacterium salinarum. This complex consists of a retinal cofactor and bacterioopsin (BO), the BR apoprotein. To examine possible novel regulatory mechanisms linking BO and retinal biosynthesis, we deleted bop, the gene that encodes BO. bop deletion resulted in a dramatic increase of bacterioruberins, carotenoid molecules that share biosynthetic precursors with retinal. Additional studies revealed that bacterioruberins accumulate in the absence of BO regardless of the presence of retinal or BR, suggesting that BO inhibits bacterioruberin biosynthesis to increase the availability of carotenoid precursors for retinal biosynthesis. To further examine this potential regulatory mechanism, we characterized an enzyme, encoded by the lye gene, that catalyzes bacterioruberin biosynthesis. BO-mediated inhibition of bacterioruberin synthesis appears to be specific to the H. salinarum lye-encoded enzyme, as expression of a lye homolog from Haloferax volcanii, a related archaeon that synthesizes bacterioruberins but lacks opsins, resulted in bacterioruberin synthesis that was not reduced in the presence of BO. Our results provide evidence for a novel regulatory mechanism in which biosynthesis of a cofactor is promoted by apoprotein-mediated inhibition of an alternate biochemical pathway. Specifically, BO accumulation promotes retinal production by inhibiting bacterioruberin biosynthesis.     

Synthesis and Properties of Bacteriorhodopsin Analogs Containing Electron-Density Labels in the Chromophore Moiety

A method for synthesis of retinal analogs labeled with electron-density groups is suggested. The interaction of these polyene compounds with bacterioopsin in apomembrane of Halobacterium salinarum was tested. A retinal analog containing a crown-ether receptor group is able to interact readily with bacterioopsin giving rise to rapid formation of a pigment with absorption maximum at 460 nm. This pigment is capable of undergoing cyclic photoconversion. The crown-bacteriorhodopsin photocycle is extremely slow and its quantum efficiency is very low (3% of that in native bacteriorhodopsin). This photocycle includes an M-like intermediate with a differential absorption maximum at 380 nm. A retinal analog in which the -ionone ring is replaced by ferrocene moiety forms a stable chromoprotein with the main absorption band at 483 nm and a shoulder near 590-610 nm.     

细菌视紫红质中质子泵出的分子机理

细菌视紫红质(Bacteriorhodopsin,或bR)是盐生嗜盐菌(Halobacterium salinarium)等细菌的跨膜蛋白质,其色基视黄醛的光致异构化作用触发细菌视紫红质的一系列结构变化,把质子从细胞质泵到细胞外空间。对细菌视紫红质中质子泵出分子机理进行了描述。     

Analysis of conformational changes in bacteriorhodopsin upon retinal removal.

The conformation of bacterioopsin in the apomembrane has been studied by Fourier transform infrared spectroscopy. Resolution enhancement techniques and curve-fitting procedures have been used to determine the secondary structural components from the amide I region. Bacterioopsin contains about 54% helicoidal structure (alpha I and alpha II helices + 3(10) turns), 21% sheets, 16% reverse turns, and 9% unordered structure. Thus, after retinal removal, all of the secondary structural types of bacteriorhodopsin remain present, and only slight quantitative differences appear. On the other hand, H/D exchange studies show that there is a higher degree of exchange for reverse turns and protonated carboxylic lateral chains in bacterioopsin as compared to bacteriorhodopsin. This gives further support to the idea of a more open tertiary structure of bacterioopsin, and to the consideration of the retinal molecule as an important element in complementing the interhelical interactions in bacteriorhodopsin folding.     

The chromophore retinal hinders passive proton/hydroxide ion translocation through bacteriorhodopsin

Experiments have been performed to examine any influence of the chromophore retinal in bacteriorhodopsin (BR) on the passive proton/hydroxide ion flux through this integral membrane protein. BR was reconstituted into dimyristoylphosphatidylcholine (DMPC)-phosphatidylserine or DMPC-dimyristoylphosphatidylglycerol unilamellar vesicles with molar lipid to protein ratios ranging from 30 to 150. The entrapped fluorescence dye pyranine served as a reliable indicator of the internal proton concentration. Transmembrane pH-gradients were quickly established across the vesicular membrane and the kinetics of the induced fluorescence changes were compared for vesicles with incorporated native BR, BR bleached to the chromophore-free protein bacterioopsin, and BR regenerated from bacterioopsin with all-trans-retinal, respectively. For aggregated protein molecules, the H+/OH- diffusion across bacterioopsin was always considerably faster than that through the protein containing covalently bound retinal. The decay rate of the imposed pH-gradient was 4.4-9.1 and 2.0-5.1 times slower for native and regenerated BR, respectively, as compared to bacterioopsin. Stepwise regeneration of bacterioopsin with all-trans-retinal revealed a linear dependence of the predominant delta pH-decay time on the degree of regeneration. Essentially the same observations were made with monomeric protein molecules in vesicular lipid membranes. The results demonstrate that the chromophore retinal itself blocks the H+/OH- conducting pathway across the transmembrane protein BR or indirectly controls this path by inducing conformational changes in the protein upon binding.     

Retinal location in purple membrane of Halobacterium halobium: a neutron diffraction study of membranes labelled in vivo with deuterated retinal

Purple membranes were prepared by growing Halobacterium halobium in a medium containing nicotine (which inhibits biosynthesis of retinal) and the oxidation products of fully deuterated beta-carotene. This allowed the in vivo incorporation of deuterated retinal into the membranes. The labelled membranes were crystalline and isomorphous with native membrane as determined by X-ray diffraction, and their optical absorption spectra were very similar. Neutron diffraction data for the two dimensional in-plane lattice from labelled and native membranes were analysed by difference Fourier and direct methods to 8.6 A resolution. The difference Fourier shows the retinal to be located in the centre of the bacteriorhodopsin molecule. The best fit to the data was obtained with the projection of retinal as a 10 A long rod forming an angle of -40 degrees +/- 10 degrees with the x axis centred at x = -0.19 +/- 0.02, y = -0.35 +/- 0.02 in fractional unit cell coordinates. The main peak in the difference Fourier map is at x = -0.17, y = -0.33.     

Evidence for a carboxyl group in the vicinity of the retinal chromophore of bacteriorhodopsin

Carboxyl groups of bacteriorhodopsin in purple membranes were activated using a hydrophobic reagent and then covalently labeled with a pH-sensitive reporter group, nitrotyrosine methyl ester. The membrane-bound reporter group had different spectral properties, and a pK 3 units higher than in solution. In purple membranes, an isosbestic point between the 428nm absorption peak of nitrotyrosine methyl ester, and the bacteriorhodopsin 570nm chromophore seen in alkaline titration, indicated interactions between the reporter group and retinal. Modification of white membranes (bacterioopsin from R₁mW strain) revealed similar, unusual spectral and ionization properties. Thus, the hydrophobic environment, not retinal interactions $\text{per}\text{se}$, are responsible for the ionization behavior of the reporter group. These results indicate that a carboxyl group is near the retinal chromophore of bacteriorhodopsin.     

Characterization of the chromophore of the third rhodopsin-like pigment of Halobacterium halobium and its photoproduct.

Halobacterium halobium contains at least three retinal-containing pigments: bacteriorhodopsin, halorhodopsin, and a third rhodopsin-like pigment (tR) absorbing at approximately 590 nm, tR590. Illumination of tR590 gives rise to a very long-lived blue absorbing photoproduct, tR370. Using high-performance liquid chromatography we show that the chromophore of tR590 is primarily all-trans retinal and its conversion by light to tR370 causes the chromophore to isomerize primarily to the 13-cis conformation. Irradiation of the tR370 gives rise to a transient photoproduct absorbing at approximately 520 nm that decays back to the initial pigment tR590. In addition to all-trans retinal, the apomembrane of tR can also combine with 13-cis retinal but not with the 9- or 11-cis isomers.     

An Australian halobacterium contains a novel proton pump retinal protein: archaerhodopsin

A bacterial strain collected from Western Australia carries all the specific features of Halobacteria, an extremely halophilic archaebacteria. This strain, Halobacterium sp. aus-1, contains a retinal protein which does differ from bacteriorhodopsin but still pumps out protons in the light. This novel proton pump is named "archaerhodopsin".