Iron-sulfur cluster-dependent catalysis of chlorophyllide a oxidoreductase from Roseobacter denitrificans.

Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)2 with the catalytic (BchY/BchZ)2 protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF4(-). Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)2. A second [4Fe-4S] cluster was identified on (BchY/BchZ)2. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.

Recoding the genetic code with selenocysteine.

Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as the Sec insertion machinery was found to be able to site-specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid (AA) to Sec was observed along with a tenfold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high-fidelity sense-codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl-tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell.

Crystal structure of the nitrogenase-like dark operative protochlorophyllide oxidoreductase catalytic complex (ChlN/ChlB)2.

During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL(2) bearing an intersubunit [4Fe-4S] cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)(2) complex. The latter contains two intersubunit [4Fe-4S] clusters and two protochlorophyllide binding sites, respectively. Here we present the crystal structure of the catalytic (ChlN/ChlB)(2) complex of DPOR from the cyanobacterium Thermosynechococcus elongatus at a resolution of 2.4 A. Subunits ChlN and ChlB exhibit a related architecture of three subdomains each built around a central, parallel beta-sheet surrounded by alpha-helices. The (ChlN/ChlB)(2) crystal structure reveals a [4Fe-4S] cluster coordinated by an aspartate oxygen alongside three cysteine ligands. Two equivalent substrate binding sites enriched in aromatic residues for protochlorophyllide substrate binding are located at the interface of each ChlN/ChlB half-tetramer. The complete octameric (ChlN/ChlB)(2)(ChlL(2))(2) complex of DPOR was modeled based on the crystal structure and earlier functional studies. The electron transfer pathway via the various redox centers of DPOR to the substrate is proposed.

Biosynthesis of (bacterio)chlorophylls: ATP-dependent transient subunit interaction and electron transfer of dark operative protochlorophyllide oxidoreductase.

Dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent two-electron reduction of protochlorophyllide a to form chlorophyllide a, the last common precursor of chlorophyll a and bacteriochlorophyll a biosynthesis. During ATP-dependent DPOR catalysis the homodimeric ChlL(2) subunit carrying a [4Fe-4S] cluster transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlN/ChlB)(2), which also possesses a redox active [4Fe-4S] cluster. To investigate the transient interaction of both subcomplexes and the resulting electron transfer reactions, the ternary DPOR enzyme holocomplex comprising subunits ChlN, ChlB, and ChlL from the cyanobacterium Prochlorococcus marinus was trapped as an octameric (ChlN/ChlB)(2)(ChlL(2))(2) complex after incubation with the nonhydrolyzable ATP analogs adenosine 5'-(gamma-thio)triphosphate, adenosine 5'-(beta,gamma-imido)triphosphate, or MgADP in combination with AlF(4)(-). Additionally, a mutant ChlL(2) protein, with a deleted Leu(153) in the switch II region also allowed for the formation of a stable octameric complex. Furthermore, efficient complex formation required the presence of protochlorophyllide. Electron paramagnetic resonance spectroscopy of ternary DPOR complexes revealed a reduced [4Fe-4S] cluster located on ChlL(2), indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer. Circular dichroism spectroscopic experiments indicated nucleotide-dependent conformational changes for ChlL(2) after ATP binding. A nucleotide-dependent switch mechanism triggering ternary complex formation and electron transfer was concluded. From these results a detailed redox cycle for DPOR catalysis was deduced.

A synthetic tRNA for EF-Tu mediated selenocysteine incorporation in vivo and in vitro.

Incorporation of selenocysteine (Sec) in bacteria requires a UGA codon that is reassigned to Sec by the Sec-specific elongation factor SelB and a conserved mRNA motif (SECIS element). These requirements severely restrict the engineering of selenoproteins. Earlier, a synthetic tRNASec was reported that allowed canonical Sec incorporation by EF-Tu; however, serine misincorporation limited its scope. We report a superior tRNASec variant (tRNAUTuX) that facilitates EF-Tu dependent stoichiometric Sec insertion in response to UAG both in vivo in Escherichia coli and in vitro in a cellfree protein synthesis system. We also demonstrate recoding of several sense codons in a SelB supplemented cell-free system. These advances in Sec incorporation will aid rational design and directed evolution of selenoproteins.

Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active-site formation and catalytic specificity.

The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA(Sec)). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA(Sec), formed by seryl-tRNA synthetase, to Sec-tRNA(Sec). SelA, a member of the fold-type-I pyridoxal 5'-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA(Sec) revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA(Sec). The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of "depentamerized" SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5'-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.

Decameric SelA•tRNA(Sec) ring structure reveals mechanism of bacterial selenocysteine formation.

The 21st amino acid, selenocysteine (Sec), is synthesized on its cognate transfer RNA (tRNA(Sec)). In bacteria, SelA synthesizes Sec from Ser-tRNA(Sec), whereas in archaea and eukaryotes SepSecS forms Sec from phosphoserine (Sep) acylated to tRNA(Sec). We determined the crystal structures of Aquifex aeolicus SelA complexes, which revealed a ring-shaped homodecamer that binds 10 tRNA(Sec) molecules, each interacting with four SelA subunits. The SelA N-terminal domain binds the tRNA(Sec)-specific D-arm structure, thereby discriminating Ser-tRNA(Sec) from Ser-tRNA(Ser). A large cleft is created between two subunits and accommodates the 3'-terminal region of Ser-tRNA(Sec). The SelA structures together with in vivo and in vitro enzyme assays show decamerization to be essential for SelA function. SelA catalyzes pyridoxal 5'-phosphate-dependent Sec formation involving Arg residues nonhomologous to those in SepSecS. Different protein architecture and substrate coordination of the bacterial enzyme provide structural evidence for independent evolution of the two Sec synthesis systems present in nature.

Enzymatic systems with homology to nitrogenase.

Nitrogenase-like dark operative protochlorophyllide oxidoreductase (DPOR) is involved in the two-electron reduction of protochlorophyllide to form chlorophyllide during chlorophyll biosynthesis. Formation of bacteriochlorophyll additionally requires a structurally related enzyme system which is termed chlorophyllide oxidoreductase (COR). During DPOR catalysis, the homodimeric subunit ChlL(2) transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlN/ChlB)(2). Analogously, subunit BchX(2) of the COR enzymes delivers electrons to subunit (BchY/BchZ)(2). The ChlL(2) protein is a dynamic switch protein triggering the ATP-dependent transfer of electrons via a [4Fe-4S] cluster onto a second [4Fe-4S] cluster located on subunit (ChlN/ChlB)(2). This initial electron transfer step of DPOR catalysis clearly resembles nitrogenase catalysis. However, the subsequent substrate reduction process was proposed to be unrelated since no molybdenum-containing cofactor or a P-cluster equivalent is employed. To investigate the transient interaction of both subcomplexes ChlL(2) and (ChlN/ChlB)(2) and the resulting electron transfer processes, the ternary DPOR enzyme holocomplex was trapped as an octameric (ChlN/ChlB)(2)(ChlL(2))(2) complex after incubation with non-hydrolyzable ATP analogs. Electron paramagnetic resonance spectroscopic experiments of various DPOR complexes in combination with circular dichroism spectroscopic experiments of the ChlL(2) protein revealed a detailed redox catalytic cycle for nucleotide-dependent DPOR catalysis.

Methods for nitrogenase-like dark operative protochlorophyllide oxidoreductase.

Nitrogenase-like dark operative protochlorophyllide oxidoreductase (DPOR) is involved in the biosynthesis of chlorophylls and bacteriochlorophylls in gymnosperms, ferns, algae, and photosynthetic bacteria. During protochlorophyllide (Pchlide) reduction, the homodimeric subunit ChlL(2) of DPOR transfers electrons on the corresponding heterotetrameric catalytic subunit (ChlN/ChlB)(2). Although DPOR shares significant amino acid sequence homology to the nitrogenase system, only the initial catalytic steps of DPOR resemble nitrogenase catalysis. Investigation of the cyanobacterial DPOR from Prochlorococcus marinus indicated that subcomplex ChlL(2) is functioning as an ATP-dependent switch protein, triggering the transient interaction of ChlL(2) and (ChlN/ChlB)(2). This dynamic subunit interplay is responsible for the transfer of a single electron from the [4Fe-4S] cluster of ChlL(2) onto a second [4Fe-4S] cluster located on (ChlN/ChlB)(2). However, the second part of DPOR catalysis is unrelated to nitrogenase catalysis, since no molybdenum-containing cofactor or a P-cluster equivalent is employed. Instead, two consecutive electron transfer steps are mediated via the [4Fe-4S] cluster of (ChlN/ChlB)(2), resulting in the reduction of the conjugated ring system of the substrate molecule Pchlide (Figs. 5.1a and 5.2).

Protochlorophyllide: a new photosensitizer for the photodynamic inactivation of Gram-positive and Gram-negative bacteria.

The growing resistance against antibiotics demands the search for alternative treatment strategies. Photodynamic therapy is a promising candidate. The natural intermediate of chlorophyll biosynthesis, protochlorophyllide, was produced, purified and tested as a novel photosensitizer for the inactivation of five model organisms including Staphylococcus aureus, Listeria monocytogenes and Yersinia pseudotuberculosis, all responsible for serious clinical infections. When microorganisms were exposed to white light from a tungsten filament lamp (0.1 mW cm(-2)), Gram-positive S. aureus, L. monocytogenes and Bacillus subtilis were photochemically inactivated at concentrations of 0.5 mg L(-1) protochlorophyllide. Transmission electron microscopy revealed a disordered septum formation during cell division and the partial loss of the cytoplasmic cell contents. Gram-negative Y. pseudotuberculosis and Escherichia coli were found to be insensitive to protochlorophyllide treatment due to the permeability barrier of the outer membrane. However, the two bacteria were rendered susceptible to eradication by protochlorophyllide (10 mg L(-1)) upon addition of polymyxin B nonapeptide at 50 and 20 mg L(-1), respectively. The release of DNA and a detrimental rearrangement of the cytoplasm were observed.

Chimeric nitrogenase-like enzymes of (bacterio)chlorophyll biosynthesis.

Nitrogenase-like light-independent protochlorophyllide oxidoreductase (DPOR) is involved in chlorophyll biosynthesis. Bacteriochlorophyll formation additionally requires the structurally related chlorophyllide oxidoreductase (COR). During catalysis, homodimeric subunit BchL(2) or ChlL(2) of DPOR transfers electrons to the corresponding heterotetrameric catalytic subunit, (BchNB)(2) or (ChlNB)(2). Analogously, subunit BchX(2) of the COR enzymes delivers electrons to subunit (BchYZ)(2). Various chimeric DPOR enzymes formed between recombinant subunits (BchNB)(2) and BchL(2) from Chlorobaculum tepidum or (ChlNB)(2) and ChlL(2) from Prochlorococcus marinus and Thermosynechococcus elongatus were found to be enzymatically active, indicating a conserved docking surface for the interaction of both DPOR protein subunits. Biotin label transfer experiments revealed the interaction of P. marinus ChlL(2) with both subunits, ChlN and ChlB, of the (ChlNB)(2) tetramer. Based on these findings and on structural information from the homologous nitrogenase system, a site-directed mutagenesis approach yielded 10 DPOR mutants for the characterization of amino acid residues involved in protein-protein interaction. Surface-exposed residues Tyr(127) of subunit ChlL, Leu(70) and Val(107) of subunit ChlN, and Gly(66) of subunit ChlB were found essential for P. marinus DPOR activity. Next, the BchL(2) or ChlL(2) part of DPOR was exchanged with electron-transferring BchX(2) subunits of COR and NifH(2) of nitrogenase. Active chimeric DPOR was generated via a combination of BchX(2) from C. tepidum or Roseobacter denitrificans with (BchNB)(2) from C. tepidum. No DPOR activity was observed for the chimeric enzyme consisting of NifH(2) from Azotobacter vinelandii in combination with (BchNB)(2) from C. tepidum or (ChlNB)(2) from P. marinus and T. elongatus, respectively.

ATP-driven reduction by dark-operative protochlorophyllide oxidoreductase from Chlorobium tepidum mechanistically resembles nitrogenase catalysis.

During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.15 nmol min(-1) mg(-1) with K(m) values of 6.1 microm for protochlorophyllide, 13.5 microm for ATP, and 52.7 microm for the reductant dithionite. To identify residues important in DPOR function, 21 enzyme variants were generated by site-directed mutagenesis and investigated for their metal content, spectroscopic features, and catalytic activity. Two cysteine residues (Cys(97) and Cys(131)) of homodimeric BchL(2) are found to coordinate an intersubunit [4Fe-4S] cluster, essential for low potential electron transfer to (BchNB)(2) as part of the reduction of the protochlorophyllide substrate. Similarly, Lys(10) and Leu(126) are crucial to ATP-driven electron transfer from BchL(2). The activation energy of DPOR electron transfer is 22.2 kJ mol(-1) indicating a requirement for 4 ATP per catalytic cycle. At the amino acid level, BchL is 33% identical to the nitrogenase subunit NifH allowing a first tentative structural model to be proposed. In (BchNB)(2), we find that four cysteine residues, three from BchN (Cys(21), Cys(46), and Cys(103)) and one from BchB (Cys(94)), coordinate a second inter-subunit [4Fe-4S] cluster required for catalysis. No evidence for any type of molybdenum-containing cofactor was found, indicating that the DPOR subunit BchN clearly differs from the homologous nitrogenase subunit NifD. Based on the available data we propose an enzymatic mechanism of DPOR.

Substrate recognition of nitrogenase-like dark operative protochlorophyllide oxidoreductase from Prochlorococcus marinus.

Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ringDbya protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR plays an important role in chlorophyll biosynthesis of gymnosperms, mosses, ferns, algae, and photosynthetic bacteria in the absence of light. Although DPOR shares significant amino acid sequence homologies with nitrogenase, only the initial catalytic steps resemble nitrogenase catalysis. Substrate coordination and subsequent [Fe-S] cluster-dependent catalysis were proposed to be unrelated. Here we characterized the first cyanobacterial DPOR consisting of the homodimeric protein complex ChlL(2) and a heterotetrameric protein complex (ChlNB)(2). The ChlL(2) dimer contains one EPR active [4Fe-4S] cluster, whereas the (ChlNB)(2) complex exhibited EPR signals for two [4Fe-4S] clusters with differences in their g values and temperature-dependent relaxation behavior. These findings indicate variations in the geometry of the individual [4Fe-4S] clusters found in (ChlNB)(2). For the analysis of DPOR substrate recognition, 11 synthetic derivatives with altered substituents on the four pyrrole rings and the isocyclic ring plus eight chlorophyll biosynthetic intermediates were tested as DPOR substrates. Although DPOR tolerated minor modifications of the ring substituents on rings A-C, the catalytic target ring D was apparently found to be coordinated with high specificity. Furthermore, protochlorophyllide a, the corresponding [8-vinyl]-derivative and protochlorophyllide b were equally utilized as substrates. Distinct differences from substrate binding by LPOR were observed. Alternative biosynthetic routes for cyanobacterial chlorophyll biosynthesis with regard to the reduction of the C8-vinyl group and the interconversion of a chlorophyll a/b type C7 methyl/formyl group were deduced.