Quenching of chlorophyll a fluorescence in the aggregates of LHCII: steady state fluorescence and picosecond relaxation kinetics

The protein composition, steady state and time-resolved fluorescence emission spectra were studied in solubilized and aggregated LHCII complexes, that were prepared according to two different isolation protocols: (1) by fractionation of cation-depleted thylakoid membranes using the non-ionic detergent Triton X-100 according to the procedure of Burke et al. [(1978) Arch. Biochem. Biophys. 187, 252-263] or (2) by solubilization with N-beta-dodecyl maltoside (beta-DM) of photosystem II (PSII) membrane fragments in the presence of cations [Irrgang et al. (1988) Eur. J. Biochem. 178, 207-217]. Based on the analysis of the decay-associated emission spectra measured at 10 and 80 K five long-wavelength chlorophyll species were identified in aggregated LHCII complexes. These five forms are characterized by emission maxima at 681.5, 683, 687, 695, or 702 nm. All of these forms were found in both types of LHCII preparations but the relative amounts and temperature dependency of these species were markedly different in the aggregated LHCII complexes isolated by the two procedures. It was found that these differences cannot be simply explained by effects due to using a less mild detergent as beta-DM or by an ionic influence of Ca2+. Biochemical analysis of the protein composition showed that beta-DM type LHCII consists of all the chlorophyll (Chl)binding proteins belonging to the antenna system of PSII except the CP29 type II gene product (CP29). In contrast, the Triton X-100-solubilized LHCII is highly depleted in CP26 (CP 29 type I gene product) and is contaminated by a variety of unidentified polypeptides. It is proposed that the aggregates of LHCII prepared using Triton X-100 acquire specific spectral and kinetic features due to interaction between the bulk of LHCII subunits and minor protein(s).     

Chloroplast SRP54 interacts with a specific subset of thylakoid precursor proteins

Signal recognition particles (SRPs) have been identified in organisms as diverse as mycoplasma and mammals; in several cases these SRPs have been shown to play a key role in protein targeting. In each case the recognition of appropriate targeting signals is mediated by SRP subunits related to the 54-kDa protein of mammalian SRP (SRP54). In this study we have characterized the specificity of 54CP, a chloroplast homologue of SRP54 which is located in the chloroplast stroma. We have used a nascent chain cross-linking approach to detect the interactions of 54CP with heterologous endoplasmic reticulum-targeting signals. 54CP functions as a bona fide signal recognition factor which can discriminate between functional and non-functional targeting signals. Using a range of authentic thylakoid precursor proteins we found that 54CP discriminates between thylakoid-targeting signals, interacting with only a subset of protein precursors. Thus, the light-harvesting chlorophyll a/b-binding protein, cytochrome f, and the Rieske FeS protein all showed strong cross-linking products with 54CP. In contrast, no cross-linking to the 23- and 33-kDa proteins of the oxygen-evolving complex were detected. The selectivity of 54CP correlates with the hydrophobicity of the thylakoid-targeting signal and, in the case of light-harvesting chlorophyll a/b-binding protein, with previously determined transport/integration requirements. We propose that 54CP mediates the targeting of a specific subset of precursors to the thylakoid membrane, i.e. those with particularly hydrophobic signal sequences.     

Light-harvesting chlorophyll a/b-binding protein stably inserts into etioplast membranes supplemented with Zn-pheophytin a/b

Light-harvesting chlorophyll a/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-pheophytin a/b, synthesized in situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Zn-pheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Zn-pheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll- or Zn-pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll- or Zn-pheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.     

Molecular cloning and functional expression of a water-soluble chlorophyll protein, a putative carrier of chlorophyll molecules in cauliflower

A cDNA for a water-soluble chlorophyll (Chl) protein (WSCP) from cauliflower (Brassica oleracea L. var botrys) was cloned and sequenced. The cDNA contained an open reading frame encoding 19 residues for a signal peptide and 199 residues for the mature form of WSCP. The sequence showed extensive homology to drought-stress-related, 22-kDa proteins in some Brassicaceae plants. Functional WSCP was expressed in Escherichia coli as a fusion protein with a maltose-binding protein (MBP). When the recombinant MBP-WSCP was incubated with thylakoid membranes, the MBP-WSCP removed Chls from these membranes. During this process, the monomer of the apo-MBP-WSCP successfully bound Chls and was converted into tetrameric holo-MBP-WSCP. The reconstituted MBP-WSCP exhibited absorption and fluorescent spectra identical to those of the native WSCP purified from cauliflower leaves. The Chl a/b ratio in native WSCP indicates a high content of Chl a, which was mainly due to the higher affinity of MBP-WSCP for Chl a. WSCP is the first example of a hydrophilic protein that can transfer Chls from thylakoid hydrophobic proteins. Possible functions of WSCP are discussed.     

Effects of cations upon chloroplast membrane subunit. Interactions and excitation energy distribution

When isolated chloroplasts from mature pea (Pisum sativum) leaves were treated with digitonin under "low salt" conditions, the membranes were extensively solubilized into small subunits (as evidenced by analysis with small pore ultrafilters). From this solubilized preparation, a photochemically inactive chlorophyll - protein complex (chlorophyll alpha/beta ratio, 1.3) was isolated. We suggest that the detergent-derived membrane fragment from mature membranes is a structural complex within the membrane which contains the light-harvesting chlorophyll alpha/beta protein and which acts as a light-harvesting antenna primarily for Photosystem II. Cations dramatically alter the structural interaction of the light-harvesting complex with the photochemically active system II complex. This interaction has been measured by determining the amount of protein-bound chlorophyll beta and Photosystem II activity which can be released into dispersed subunits by digitonin treatment of chloroplast lamellae. When cations are present to cause interaction between the Photosystem II complex and the light-harvesting pigment - protein, the combined complexes pellet as a "heavy" membranous fraction during differential centrifugation of detergent treated lamellae. In the absence of cations, the two complexes dissociate and can be isolated in a "light" submembrane preparation from which the light-harvesting complex can be purified by sucrose gradient centrifugation. Cation effects on excitation energy distribution between Photosystems I and II have been monitored by following Photosystem II fluorescence changes under chloroplast incubation conditions identical to those used for detergent treatment (with the exception of chlorophyll concentration differences and omission of detergents). The cation dependency of the pigment - protein complex and Photosystem II reaction center interactions measured by detergent fractionation, and regulation of excitation energy distribution as measured by fluorescence changes, were identical. We conclude that changes in substructural organization of intact membranes, involving cation induced changes in the interaction of intramembranous subunits, are the primary factors regulating the distribution of excitation energy between Photosystems II and I.     

New Arabidopsis cue mutants suggest a close connection between plastid- and phytochrome regulation of nuclear gene expression

We searched for new components that are involved in the positive regulation of nuclear gene expression by light by extending a screen for Arabidopsis cue (chlorophyll a/b-binding [CAB] protein-underexpressed) mutants (H.-M. Li, K. Culligan, R.A. Dixon, J. Chory [1995] Plant Cell 7: 1599-1610). cue mutants display reduced expression of the CAB3 gene, which encodes light-harvesting chlorophyll protein, the main chloroplast antenna. The new mutants can be divided into (a) phytochrome-deficient mutants (hy1 and phyB), (b) virescent or delayed-greening mutants (cue3, cue6, and cue8), and (c) uniformly pale mutants (cue4 and cue9). For each of the mutants, the reduction in CAB expression correlates with the visible phenotype, defective chloroplast development, and reduced abundance of the light-harvesting chlorophyll protein. Levels of protochlorophyllide oxidoreductase (POR) were reduced to varying degrees in etiolated mutant seedlings. In the dark, whereas the virescent mutants displayed reduced CAB expression and the lowest levels of POR protein, the other mutants expressed CAB and accumulated POR at near wild-type levels. All of the mutants, with the exception of cue6, were compromised in their ability to derepress CAB expression in response to phytochrome activation. Based on these results, we propose that the previously postulated plastid-derived signal is closely involved in the pathway through which phytochrome regulates the expression of nuclear genes encoding plastid proteins.     

Biochemical and functional properties of photosystem II in agranal membranes from maize mesophyll and bundle sheath chloroplasts

We have studied the occurrence and organization of photosystem II (PSII) in bundle sheath thylakoids and stroma lamellae from maize. As shown by non-denaturing lauryl beta-D- iminopropionidate (Deriphat)/PAGE, PSII exists in a dimeric form in grana membranes. In bundle sheath and stroma lamellae, however, only a monomeric form was found. Based on immunotitration data, we estimated the stoichiometry of the individual components of the PSII core complex and antenna systems. In stroma lamellae, all PSII antenna complexes had a stoichiometry similar to that in grana membranes, with the exception of light-harvesting complex II (LHCII) that was somewhat over-represented, while the minor antenna complexes CP26 and CP29 were under-represented. In bundle sheath, the amount of LHCII was approximately eight times higher than expected with respect to D1. The 33-kDa protein of the oxygen-evolving enhancer polypeptides was not detectable nor was the ferredoxin-NADP+ reductase, thus strongly suggesting that no significant linear electron transport occurs in bundle sheath thylakoids. Fluorescence induction data suggest that most of the PSII reaction centers in bundle sheath and stroma lamellae sustain electron transport towards a secondary acceptor pool. Stromal PSII centers are only weakly inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), whereas, unexpectedly, dichlorobenzoquinone and methyl viologen had a pronounced inhibitory effect of the QA- reoxidation. An additional specificity of these centers is the slow rate (50-ms range) of the QA to QB electron transfer. The amplitude of variable fluorescence found in stroma lamellae can only account for a small fraction (1-2%) of the variable fluorescence of whole thylakoids. This suggests that stromal PSII cannot be solely responsible for the slow beta-phase of the induction kinetics.     

Ultrafast evolution of the excited states in the chlorophyll a/b complex CP29 from green plants studied by energy-selective pump-probe spectroscopy

The energy transfer process in the minor light-harvesting antenna complex CP29 of green plants was probed in multicolor transient absorption experiments at 77 K using selective subpicosecond excitation pulses at 640 and 650 nm. Energy flow from each of the chlorophyll (Chl) b molecules of the complex could thus be studied separately. The analysis of our data showed that the "blue" Chl b (absorption around 640 nm) transfers excitation to a "red" Chl a with a time constant of 350 +/- 100 fs, while the 'red' Chl b (absorption at 650 nm) transfers on a picosecond time scale (2.2 +/- 0.5 ps) toward a "blue" Chl a. Furthermore, both fast (280 +/- 50 fs) and slow (10-13 ps) equilibration processes among the Chl a molecules were observed, with rates and associated spectra very similar to those of the major antenna complex, LHC-II. Based on the protein sequence homology between CP29 and LHC-II, a basic modelling of the observed kinetics was performed using the LHC-II structure and the F?rster theory of energy transfer. Thus, an assignment for the spectral properties and orientation of the two Chl's b, as well as for their closest Chl a neighbors, is put forward, and a comparison is made with the previous assignments and models for LHC-II and CP29.     

Heterogenous lipid distribution among chlorophyll-binding proteins of photosystem II in maize mesophyll chloroplasts

Photosystem II membrane fractions from dark-adapted mesophyll chloroplasts of maize were solubilized in different concentrations of dodecyl beta-D-maltoside. Chlorophyll-binding proteins from photosystem II were isolated either by ultracentrifugation on a sucrose gradient, or by flat bed isoelectric focusing and identified by gel electrophoresis analysis for their polypeptide composition. Lipid and fatty acid compositions were determined in complexes prepared by both methods and also in purified light-harvesting complex II, in minor chlorophyll a/b binding complexes 29, 26, 24, in photosystem II antennae (chlorophyll-protein complexes 43, 47) and in the photosystem II reaction centers chlorophyll-protein complexes. Comparative analysis of the results suggests that a true heterogeneity exists in the lipid class distribution among the different chlorophyll-protein complexes in this region of the photosynthetic membrane. Photosystem II core fractions prepared either by ultra-centrifugation on a sucrose gradient or by isoelectric focusing were found significantly enriched in monogalactosyldiacylglycerol; fractionation of the photosystem II core in its components showed that it was the chlorophyll-protein complexes 43 and 47 which were mainly responsible for this enrichment. One of them, the chlorophyll-protein complex 47, was found containing monogalactosyldiacylglycerol and having a very high level of saturated fatty acids. The minor chlorophyll a/b binding linkers (chlorophyll-protein complexes 24, 26 and 29) retain a largely higher amount of lipids than all other complexes and especially of highly unsaturated galactolipids. Concerning the main light-harvesting antenna (LHCII), it is demonstrated that phosphatidylglycerol is strongly linked to the complex if it cannot be detached at high detergent concentration, while many galactolipids (which nevertheless represent the major lipid classes) are lost. This main light-harvesting complex has been fractionated into several families by isoelectric focusing showing a marked difference in lipid and polypeptide composition. A spectacular increase in the phosphatidylglycerol content was observed in the fraction migrating near the anode and enriched in a 26-kDa polypeptide; but this result is difficult to interpret in physiological terms as it was shown that phosphatidylglycerol alone, because of its negative charge, also migrates toward the anode in isoelectric focusing.     

Energy transfer in LHCII monomers at 77K studied by sub-picosecond transient absorption spectroscopy

Energy transfer from chlorophyll b (Chl b) to chlorophyll a (Chl a) in monomeric preparations of light-harvesting complex II (LHCII) from spinach was studied at 77 K using pump-probe experiments. Sub-picosecond excitation pulses centered at 650 nm were used to excite preferentially Chl b and difference absorption spectra were detected from 630 to 700 nm. Two distinct Chl b to Chl a transfer times, approximately 200 fs and 3 ps, were found. A clearly distinguishable energy transfer process between Chl a molecules occurred with a time constant of 18 ps. The LHCII monomer data are compared to previously obtained LHCII trimer data, and both data sets are fitted simultaneously using a global analysis fitting routine. Both sets could be described with the following time constants: 140 fs, 600 fs, 8 ps, 20 ps, and 2.9 ns. In both monomers and trimers 50% of the Chl b to Chl a transfer is ultrafast (<200 fs). However, for monomers this transfer occurs to Chl a molecules that absorb significantly more toward shorter wavelengths than for trimers. Part of the transfer from Chl b to Chl a that occurs with a time constant of 600 fs in trimers is slowed down to several picoseconds in monomers. However, it is argued that observed differences between monomers and trimers should be ascribed to the loss of some Chl a upon monomerization or a shift of the absorption maximum of one or several Chl a molecules. It is concluded that Chl b to Chl a transfer occurs only within monomeric subunits of the trimers and not between different subunits.     

Characterization of a 2[4Fe-4S] ferredoxin obtained by chemical insertion of the Fe-S clusters into the apoferredoxin II from Rhodobacter capsulatus

The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one [3Fe-4S] cluster and one [4Fe-4S] cluster. This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin. The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe-S clusters under anaerobic conditions. A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms. The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH. The reconstituted ferredoxin was purified and shown to be distinct from the native [7Fe-8S] ferredoxin, based on several biochemical and spectroscopic criteria. In the oxidized state, EPR revealed the quasi-absence of [3Fe-4S] cluster. 1H-NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2[4Fe-4S] ferredoxin. This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur. Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe-S clusters, without formation of [7Fe-8S] form. Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron-transfer assay coupled to nitrogenase activity. The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins.     

Reconstitution of recombinant N-formyl chemotactic peptide receptor with G protein

A recombinant human neutrophil N-formyl peptide receptor (rFPR) expressed in transfected mouse fibroblasts (TX2 cells) was analyzed for its ability to couple physically with the heterotrimeric G protein, Gi. Immunoprecipitation of photoaffinity-labeled rFPR and endogenous neutrophil formyl peptide receptor (nFPR) with an anti-FPR peptide antibody demonstrated that the receptors were identical in both size and extent of glycosylation. Coupling of rFPR with endogenous TX2 Gi was demonstrated by coimmunoprecipitation of the two proteins with an anti-Gi antibody. Moreover, rFPR was able to form a physical complex with purified Gi in a soluble reconstitution system. We observed similar affinities of rFPR and nFPR for Gi. This report provides the first direct evidence that rFPR associates physically with Gi and provides a foundation for analysis of the G protein coupling capacity of mutant rFPRs.     

A chloroplast processing enzyme functions as the general stromal processing peptidase

A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established. We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in Escherichia coli. Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle. CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase. Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc.     

Different phototransduction kinetics of phytochrome A and phytochrome B in Arabidopsis thaliana

The kinetics of phototransduction of phytochrome A (phyA) and phytochrome B (phyB) were compared in etiolated Arabidopsis thaliana seedlings. The responses of hypocotyl growth, cotyledon unfolding, and expression of a light-harvesting chlorophyll a/b-binding protein of the photosystem II gene promoter fused to the coding region of beta-glucuronidase (used as a reporter enzyme) were mediated by phyA under continuous far-red light (FR) and by phyB under continuous red light (R). The seedlings were exposed hourly either to n min of FR followed by 60 minus n min in darkness or to n min of R, 3 min of FR (to back-convert phyB to its inactive form), and 57 minus n min of darkness. For the three processes investigated here, the kinetics of phototransduction of phyB were faster than that of phyA. For instance, 15 min R h-1 (terminated with a FR pulse) were almost as effective as continuous R, whereas 15 min of FR h-1 caused less than 30% of the effect of continuous FR. This difference is interpreted in terms of divergence of signal transduction pathways downstream from phyA and phyB.     

Rate of carotenoid triplet formation in solubilized light-harvesting complex II (LHCII) from spinach

In the present study the rate of triplet transfer from chlorophyll to carotenoids in solubilized LHCII was investigated by flash spectroscopy using laser pulses of approximately 2 ns for both pump and probe. Special attention has been paid to calibration of the experimental setup and to avoid saturation effects. Carotenoid triplets were identified by the pronounced positive peak at approximately 507 nm in the triplet-singlet difference spectra. DeltaOD (507 nm) exhibits a monoexponential relaxation kinetics with characteristic lifetimes of 2-9 micros (depending on the oxygen content) that was found to be independent of the pump pulse intensity. The rise of DeltaOD (507 nm) was resolved via a pump probe technique where an optical delay of up to 20 ns was used. A thorough analysis of these experimental data leads to the conclusion that the kinetics of carotenoid triplet formation in solubilized LHCII is almost entirely limited by the lifetime of the excited singlet state of chlorophyll but neither by the pulse width nor by the rate constant of triplet-triplet transfer. Within the experimental error the rate constant of triplet-triplet transfer from chlorophyll to carotenoids was estimated to be kTT > (0.5 ns)-1. This value exceeds all data reported so far by at least one order of magnitude. The implications of this finding are briefly discussed.     

The Oms66 (p66) protein is a Borrelia burgdorferi porin

In this study we report the purification and characterization of a 66-kDa protein, designated Oms66, for outer membrane-spanning 66-kDa protein, that functions as a porin in the outer membrane (OM) of Borrelia burgdorferi. Oms66 was purified by fast-performance liquid chromatography and exhibited an average single-channel conductance of 9.62 +/- 0.37 nS in 1 M KCl, as evidenced by 581 individual insertional events in planar lipid bilayers. Electrophysiological characterization indicated that Oms66 was virtually nonselective between cations and anions and exhibited voltage-dependent closure with multiple substates. The amino acid sequence of tryptic peptides derived from purified Oms66 was identical to the deduced amino acid sequence of p66, a previously described surface-exposed protein of B. burgdorferi. Purified Oms66 was recognized by antiserum specific for p66 and serum from rabbits immune to challenge with virulent B. burgdorferi, indicating that p66 and Oms66 were identical proteins and that Oms66/p66 is an immunogenic protein in infected rabbits. In a methodology that reduces liposomal trapping and nonspecific interactions, native Oms66 was incorporated into liposomes, confirming that Oms66 is an outer membrane-spanning protein. Proteoliposomes containing Oms66 exhibited porin activity nearly identical to that of native, purified Oms66, indicating that reconstituted Oms66 retained native conformation. The use of proteoliposomes reconstituted with Oms66 and other Oms proteins provides an experimental system for determinating the relationship between conformation, protection, and biological function of these molecules.     

Topology of Euglena chloroplast protein precursors within endoplasmic reticulum to Golgi to chloroplast transport vesicles

Euglena chloroplast protein precursors are transported as integral membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus prior to chloroplast localization. All Euglena chloroplast protein precursors have functionally similar bipartite presequences composed of an N-terminal signal peptide domain and a stromal targeting domain containing a hydrophobic region approximately 60 amino acids from the predicted signal peptidase cleavage site. Asparagine-linked glycosylation reporters and presequence deletion constructs of the precursor to the Euglena light-harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) were used to identify presequence regions translocated into the ER lumen and stop transfer membrane anchor domains. An asparagine-linked glycosylation site present at amino acid 148 of pLHCPII near the N terminus of mature LHCPII was not glycosylated in vitro by canine microsomes while an asparagine-linked glycosylation site inserted at amino acid 40 was. The asparagine at amino acid 148 was glycosylated upon deletion of amino acids 46-146, which contain the stromal targeting domain, indicating that the hydrophobic region within this domain functions as a stop transfer membrane anchor sequence. Protease protection assays indicated that for all constructs, mature LHCPII was not translocated across the microsomal membrane. Taken together with the structural similarity of all Euglena presequences, these results demonstrate that chloroplast precursors are anchored within ER and Golgi transport vesicles by the stromal targeting domain hydrophobic region oriented with the presequence N terminus formed by signal peptidase cleavage in the vesicle lumen and the mature protein in the cytoplasm.     

Interaction of pyridoxal 5'-phosphate with tryptophan-139 at the subunit interface of dimeric D-amino acid transaminase

The crystal structure of dimeric bacterial D-amino acid transaminase shows that the indole rings of the two Trp-139 side chains face each other in the subunit interface about 10 angstroms from the coenzyme, pyridoxal 5'-phosphate. To determine whether it has a role in the catalytic efficiency of the enzyme or interacts with the coenzyme, Trp-139 has been substituted by several different types of amino acids, and the properties of these recombinant mutant enzymes have been compared to the wild-type enzyme. In the native wild-type holoenzyme, the fluorescence of one of the three Trp residues per monomer is almost completely quenched, probably due to its interaction with PLP since in the native wild-type apoenzyme devoid of PLP, tryptophan fluorescence is not quenched. Upon reconstitution of this apoenzyme with PLP, the tryptophan fluorescence is quenched to about the same extent as it is in the native wild-type enzyme. The site of fluorescence quenching is Trp-139 since the W139F mutant in which Trp-139 is replaced by Phe has about the same amount of fluorescence as the wild-type enzyme. The circular dichroism spectra of the holo and the apo forms of both the wild-type and the W139F enzymes in the far-ultraviolet show about the same degree of ellipticity, consistent with the absence of extensive global changes in protein structure. Furthermore, comparison of the circular dichroism spectrum of the W139F enzyme at 280 nm with the corresponding spectral region of the wild-type enzyme suggests a restricted microenvironment for Trp-139 in the latter enzyme. The functional importance of Trp-139 is also demonstrated by the finding that its replacement by Phe, His, Pro, or Ala gives mutant enzymes that are optimally active at temperatures below that of the wild-type enzyme and undergo the E-PLP --> E-PMP transition as a function of D-Ala concentration with reduced efficiency. The results suggest that a fully functional dimeric interface with the two juxtaposed indole rings of Trp-139 is important for optimal catalytic function and maximum thermostability of the enzyme and, furthermore, that there might be energy transfer between Trp-139 and coenzyme PLP.