Two-component carnitine monooxygenase from Escherichia coli: functional characterization, inhibition and mutagenesis of the molecular interface.

Gut microbial production of trimethylamine (TMA) from l-carnitine is directly linked to cardiovascular disease. TMA formation is facilitated by carnitine monooxygenase, which was proposed as a target for the development of new cardioprotective compounds. Therefore, the molecular understanding of the two-component Rieske-type enzyme from Escherichia coli was intended. The redox cofactors of the reductase YeaX (FMN, plant-type [2Fe-2S] cluster) and of the oxygenase YeaW (Rieske-type [2Fe-2S] and mononuclear [Fe] center) were identified. Compounds meldonium and the garlic-derived molecule allicin were recently shown to suppress microbiota-dependent TMA formation. Based on two independent carnitine monooxygenase activity assays, enzyme inhibition by meldonium or allicin was demonstrated. Subsequently, the molecular interplay of the reductase YeaX and the oxygenase YeaW was addressed. Chimeric carnitine monooxygenase activity was efficiently reconstituted by combining YeaX (or YeaW) with the orthologous oxygenase CntA (or reductase CntB) from Acinetobacter baumannii. Partial conservation of the reductase/oxygenase docking interface was concluded. A structure guided mutagenesis approach was used to further investigate the interaction and electron transfer between YeaX and YeaW. Based on AlphaFold structure predictions, a total of 28 site-directed variants of YeaX and YeaW were kinetically analyzed. Functional relevance of YeaX residues Arg271, Lys313 and Asp320 was concluded. Concerning YeaW, a docking surface centered around residues Arg83, Lys104 and Lys117 was hypothesized. The presented results might contribute to the development of TMA-lowering strategies that could reduce the risk for cardiovascular disease.

Radical SAM Enzymes Involved in Tetrapyrrole Biosynthesis and Insertion.

The anaerobic biosyntheses of heme, heme d 1, and bacteriochlorophyll all require the action of radical SAM enzymes. During heme biosynthesis in some bacteria, coproporphyrinogen III dehydrogenase (CgdH) catalyzes the decarboxylation of two propionate side chains of coproporphyrinogen III to the corresponding vinyl groups of protoporphyrinogen IX. Its solved crystal structure was the first published structure for a radical SAM enzyme. In bacteria, heme is inserted into enzymes by the cytoplasmic heme chaperone HemW, a radical SAM enzyme structurally highly related to CgdH. In an alternative heme biosynthesis route found in archaea and sulfate-reducing bacteria, the two radical SAM enzymes AhbC and AhbD catalyze the removal of two acetate groups (AhbC) or the decarboxylation of two propionate side chains (AhbD). NirJ, a close homologue of AhbC, is required for propionate side chain removal during the formation of heme d 1 in some denitrifying bacteria. Biosynthesis of the fifth ring (ring E) of all chlorophylls is based on an unusual six-electron oxidative cyclization step. The sophisticated conversion of Mg-protoporphyrin IX monomethylester to protochlorophyllide is facilitated by an oxygen-independent cyclase termed BchE, which is a cobalamin-dependent radical SAM enzyme. Most of the radical SAM enzymes involved in tetrapyrrole biosynthesis were recognized as such by Sofia et al. in 2001 (Nucleic Acids Res.2001, 29, 1097-1106) and were biochemically characterized thereafter. Although much has been achieved, the challenging tetrapyrrole substrates represent a limiting factor for enzyme/substrate cocrystallization and the ultimate elucidation of the corresponding enzyme mechanisms.

Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.

Carnitine metabolism in the human gut: characterization of the two-component carnitine monooxygenase CntAB from Acinetobacter baumannii.

Bacterial formation of trimethylamine (TMA) from carnitine in the gut microbiome has been linked to cardiovascular disease. During this process, the two-component carnitine monooxygenase (CntAB) catalyzes the oxygen-dependent cleavage of carnitine to TMA and malic semialdehyde. Individual redox states of the reductase CntB and the catalytic component CntA were investigated based on mutagenesis and electron paramagnetic resonance (EPR) spectroscopic approaches. Protein ligands of the flavin mononucleotide (FMN) and the plant-type [2Fe-2S] cluster of CntB and also of the Rieske-type [2Fe-2S] cluster and the mononuclear [Fe] center of CntA were identified. EPR spectroscopy of variant CntA proteins suggested a hierarchical metallocenter maturation, Rieske [2Fe-2S] followed by the mononuclear [Fe] center. NADH-dependent electron transfer via the redox components of CntB and within the trimeric CntA complex for the activation of molecular oxygen was investigated. EPR experiments indicated that the two electrons from NADH were allocated to the plant-type [2Fe-2S] cluster and to FMN in the form of a flavin semiquinone radical. Single-turnover experiments of this reduced CntB species indicated the translocation of the first electron onto the [Fe] center and the second electron onto the Rieske-type [2Fe-2S] cluster of CntA to finally allow for oxygen activation as a basis for carnitine cleavage. EPR spectroscopic investigation of CntA variants indicated an unusual intermolecular electron transfer between the subunits of the CntA trimer via the "bridging" residue Glu-205. On the basis of these data, a redox catalytic cycle for carnitine monooxygenase was proposed.

Chimeric Interaction of Nitrogenase-Like Reductases with the MoFe Protein of Nitrogenase.

The engineering of transgenic organisms with the ability to fix nitrogen is an attractive possibility. However, oxygen sensitivity of nitrogenase, mainly conferred by the reductase component (NifH)2 , is an imminent problem. Nitrogenase-like enzymes involved in coenzyme F430 and chlorophyll biosynthesis utilize the highly homologous reductases (CfbC)2 and (ChlL)2 , respectively. Chimeric protein-protein interactions of these reductases with the catalytic component of nitrogenase (MoFe protein) did not support nitrogenase activity. Nucleotide-dependent association and dissociation of these complexes was investigated, but (CfbC)2 and wild-type (ChlL)2 showed no modulation of the binding affinity. By contrast, the interaction between the (ChlL)2 mutant Y127S and the MoFe protein was markedly increased in the presence of ATP (or ATP analogues) and reduced in the ADP state. Upon formation of the octameric (ChlL)2 MoFe(ChlL)2 complex, the ATPase activity of this variant is triggered, as seen in the homologous nitrogenase system. Thus, the described reductase(s) might be an attractive tool for further elucidation of the diverse functions of (NifH)2 and the rational design of a more robust reductase.

Enzymatic Systems with Homology to Nitrogenase: Biosynthesis of Bacteriochlorophyll and Coenzyme F430.

Enzymes with homology to nitrogenase are essential for the reduction of chemically stable double bonds within the biosynthetic pathways of bacteriochlorophyll and coenzyme F430. These tetrapyrrole-based compounds are crucial for bacterial photosynthesis and the biogenesis of methane in methanogenic archaea. Formation of bacteriochlorophyll requires the unique ATP-dependent enzyme chlorophyllide oxidoreductase (COR) for the two-electron reduction of chlorophyllide to bacteriochlorophyllide. COR catalysis is based on the homodimeric protein subunit BchX2, which facilitates the transfer of electrons to the corresponding heterotetrameric catalytic subunit (BchY/BchZ)2. By analogy to the nitrogenase system, the dynamic switch protein BchX2 contains a [4Fe-4S] cluster that triggers the ATP-driven transfer of electrons onto a second [4Fe-4S] cluster located in (BchY/BchZ)2. The subsequent substrate reduction and protonation is unrelated to nitrogenase catalysis, with no further involvement of a molybdenum-containing cofactor. The biosynthesis of the nickel-containing coenzyme F430 includes the six-electron reduction of the tetrapyrrole macrocycle of Ni2+-sirohydrochlorin a,c-diamide to Ni2+-hexahydrosirohydrochlorin a,c-diamide catalyzed by CfbC/D. The homodimeric CfbC2 subunit carrying a [4Fe-4S] cluster shows close homology to BchX2. Accordingly, parallelism for the initial ATP-driven electron transfer steps of CfbC/D was proposed. Electrons are received by the dimeric catalytic subunit CfbD2, which contains a second [4Fe-4S] cluster and carries out the saturation of an overall of three double bonds in a highly orchestrated spatial and regioselective process. Following a short introduction to nitrogenase catalysis, this chapter will focus on the recent progress toward the understanding of the nitrogenase-like enzymes COR and CfbC/D, with special emphasis on the underlying enzymatic mechanism(s).

Expression, Purification, and Activity Analysis of Chlorophyllide Oxidoreductase and Ni2+-Sirohydrochlorin a,c-Diamide Reductase.

Nitrogenase-like enzymes play a vital role in the reduction of the conjugated ring systems of diverse tetrapyrrole molecules. The biosynthesis of all bacteriochlorophylls involves the two-electron reduction of the C7-C8 double bond of the green pigment chlorophyllide, which is catalyzed by the nitrogenase-like two-component metalloenzyme chlorophyllide oxidoreductase (COR); whereas in all methanogenic microbes, another nitrogenase-like system, CfbC/D, is responsible for the sophisticated six-electron reduction of Ni2+-sirohydrochlorin a,c-diamide in the course of coenzyme F430 biosynthesis. The first part of this chapter describes the production and purification of the COR components (BchY/BchZ)2 and BchX2, the measurement of COR activity, and the trapping of the ternary COR complex; and the second part describes the strategy for obtaining homogenous and catalytically active preparations of CfbC2 and CfbD2 and a suitable method for extracting the reaction product Ni2+-hexahydrosirohydrochlorin a,c-diamide.

Virulence of Agrobacterium tumefaciens requires lipid homeostasis mediated by the lysyl-phosphatidylglycerol hydrolase AcvB.

Agrobacterium tumefaciens transfers oncogenic T-DNA via the type IV secretion system (T4SS) into plants causing tumor formation. The acvB gene encodes a virulence factor of unknown function required for plant transformation. Here we specify AcvB as a periplasmic lysyl-phosphatidylglycerol (L-PG) hydrolase, which modulates L-PG homeostasis. Through functional characterization of recombinant AcvB variants, we showed that the C-terminal domain of AcvB (residues 232-456) is sufficient for full enzymatic activity and defined key residues for catalysis. Absence of the hydrolase resulted in ~10-fold increase in L-PG in Agrobacterium membranes and abolished T-DNA transfer and tumor formation. Overproduction of the L-PG synthase gene (lpiA) in wild-type A. tumefaciens resulted in a similar increase in the L-PG content (~7-fold) and a virulence defect even in the presence of intact AcvB. These results suggest that elevated L-PG amounts (either by overproduction of the synthase or absence of the hydrolase) are responsible for the virulence phenotype. Gradually increasing the L-PG content by complementation with different acvB variants revealed that cellular L-PG levels above 3% of total phospholipids interfere with T-DNA transfer. Cumulatively, this study identified AcvB as a novel virulence factor required for membrane lipid homeostasis and T-DNA transfer.

A phosphatidic acid-binding protein is important for lipid homeostasis and adaptation to anaerobic biofilm conditions in Pseudomonas aeruginosa.

A quantitative Pseudomonas aeruginosa proteomics approach revealed increased abundance of the so-far uncharacterized protein PA3911 in anaerobic biofilms grown under conditions of the cystic fibrosis lung. Physiological relevance of ORF PA3911 was demonstrated, inter alia, using phenotype microarray experiments. The mutant strain showed increased susceptibility in the presence of antimicrobials (minocycline, nafcillin, oxacillin, chloramphenicol and thiamphenicol), enhanced twitching motility and significantly impaired biofilm formation. PA3911 is a soluble, cytoplasmic protein in P. aeruginosa In protein-lipid overlay experiments, purified PA3911 bound specifically to phosphatidic acid (PA), the central hub of phospholipid metabolism. Structure-guided site-directed mutagenesis was used to explore the proposed ligand-binding cavity of PA3911. Protein variants of Leu56, Leu58, Val69 and Leu114 were shown to impair PA interaction. A comparative shotgun lipidomics approach demonstrated a multifaceted response of P. aeruginosa to anaerobic conditions at the lipid head group and fatty acid level. Lipid homeostasis in the PA3911 mutant strain was imbalanced with respect to lysophosphatidylcholine, phosphatidylcholine and diacylglycerol under anaerobic and/or aerobic conditions. The impact of the newly identified PA-binding protein on lipid homeostasis and the related macroscopic phenotypes of P. aeruginosa are discussed.

Reduction of Chemically Stable Multibonds: Nitrogenase-Like Biosynthesis of Tetrapyrroles.

The sophisticated biochemistry of nitrogenase plays a fundamental role for the biosynthesis of tetrapyrrole molecules, acting as key components of photosynthesis and methanogenesis. Three nitrogenase-like metalloenzymes have been characterized to date. Synthesis of chlorophylls and bacteriochlorophylls involves the reduction of the C17-C18 double bond of the conjugated ring system of protochlorophyllide which is catalyzed by the multi-subunit enzyme dark operative protochlorophyllide oxidoreductase (DPOR). Subsequently, biosynthesis of all bacteriochlorophylls requires the reduction of the C7-C8 double bond by a second nitrogenase-like enzyme termed chlorophyllide oxidoreductase (COR). Mechanistically, DPOR and COR make use of a reductase component which links ATP hydrolysis to conformational changes. This dynamic switch protein is triggering the transient association between the reductase and the core catalytic protein complex, thereby facilitating the transduction of electrons via two [4Fe4S] clusters. X-ray crystallographic structural investigations in combination with biochemical experiments revealed the molecular basis of the underlying energy transduction mechanism. The unique nickel-containing tetrapyrrole cofactor F430 is located in the active site of methyl-coenzyme M reductase, which is catalyzing the final step of methane formation in methanogenic archaea. The nitrogenase-like protein NflH/NflD has been proposed to catalyze one or more ring reduction steps during the biosynthesis of F430. The present working hypothesis mirrors a DPOR and COR related enzyme mechanism of NflH/NflD. Furthermore, nfl-encoded proteins were suggested as "simplified" ancestors lying basal in the phylogenetic tree between nitrogenase and DPOR/COR.

Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum.

Violacein is a natural purple pigment of Chromobacterium violaceum with potential medical applications as antimicrobial, antiviral, and anticancer drugs. The initial step of violacein biosynthesis is the oxidative conversion of l-tryptophan into the corresponding α-imine catalyzed by the flavoenzyme l-tryptophan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-methylpropanoic acid) and a product-related (2-(1H-indol-3-ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was synthesized for subsequent kinetic and x-ray crystallographic investigations. Structures of the binary VioA·FADH2 and of the ternary VioA·FADH2·2-(1H-indol-3-ylmethyl)prop-2-enoic acid complex were resolved. VioA forms a "loosely associated" homodimer as indicated by small-angle x-ray scattering experiments. VioA belongs to the glutathione reductase family 2 of FAD-dependent oxidoreductases according to the structurally conserved cofactor binding domain. The substrate-binding domain of VioA is mainly responsible for the specific recognition of l-tryptophan. Other canonical amino acids were efficiently discriminated with a minor conversion of l-phenylalanine. Furthermore, 7-aza-tryptophan, 1-methyl-tryptophan, 5-methyl-tryptophan, and 5-fluoro-tryptophan were efficient substrates of VioA. The ternary product-related VioA structure indicated involvement of protein domain movement during enzyme catalysis. Extensive structure-based mutagenesis in combination with enzyme kinetics (using l-tryptophan and substrate analogs) identified Arg(64), Lys(269), and Tyr(309) as key catalytic residues of VioA. An increased enzyme activity of protein variant H163A in the presence of l-phenylalanine indicated a functional role of His(163) in substrate binding. The combined structural and mutational analyses lead to the detailed understanding of VioA substrate recognition. Related strategies for the in vivo synthesis of novel violacein derivatives are discussed.

Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine.

The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.

Broadened Substrate Specificity of 3-Hydroxyethyl Bacteriochlorophyllide a Dehydrogenase (BchC) Indicates a New Route for the Biosynthesis of Bacteriochlorophyll a.

Bacteriochlorophyll a biosynthesis requires formation of a 3-hydroxyethyl group on pyrrole ring A that gets subsequently converted into a 3-acetyl group by 3-vinyl bacteriochlorophyllide a hydratase (BchF) followed by 3-hydroxyethyl bacteriochlorophyllide a dehydrogenase (BchC). Heterologous overproduction of Chlorobaculum tepidum BchF revealed an integral transmembrane protein that was efficiently isolated by detergent solubilization. Recombinant C. tepidum BchC was purified as a soluble protein-NAD(+) complex. Substrate recognition of BchC was investigated using six artificial substrate molecules. Modification of the isocyclic E ring, omission of the central magnesium ion, zinc as an alternative metal ion, and a non-reduced B ring system were tolerated by BchC. According to this broadened in vitro activity, the chlorin 3-hydroxyethyl chlorophyllide a was newly identified as a natural substrate of BchC in a reconstituted pathway consisting of dark-operative protochlorophyllide oxidoreductase, BchF, and BchC. The established reaction sequence would allow for an additional new branching point for the synthesis of bacteriochlorophyll a. Biochemical and site-directed mutagenesis analyses revealed, in contrast to theoretical predictions, a zinc-independent BchC catalysis that requires NAD(+) as a cofactor. Based on these results, we are designating a new medium-chain dehydrogenase/reductase family (MDR057 BchC) as theoretically proposed from a recent bioinformatics analysis.

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.

Identification and characterization of a periplasmic aminoacyl-phosphatidylglycerol hydrolase responsible for Pseudomonas aeruginosa lipid homeostasis.

Specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine (or with lysine) was shown to render various organisms less susceptible to antimicrobial agents and environmental stresses. In this study, we make use of the opportunistic pathogen Pseudomonas aeruginosa to decode ORF PA0919-dependent lipid homeostasis. Analysis of the polar lipid content of the deletion mutant ΔPA0919 indicated significantly enlarged levels of alanyl-PG. The resulting phenotype manifested an increased susceptibility to several antimicrobial compounds when compared with the wild type. A pH-dependent PA0919 promoter located within the upstream gene PA0920 was identified. Localization experiments demonstrated that the PA0919 protein is anchored to the periplasmic surface of the inner bacterial membrane. The recombinant overproduction of wild type and several site-directed mutant proteins in the periplasm of Escherichia coli facilitated a detailed in vitro analysis of the enzymatic PA0919 function. A series of artificial substrates (p-nitrophenyl esters of various amino acids/aliphatic acids) indicated enzymatic hydrolysis of the alanine, glycine, or lysine moiety of the respective ester substrates. Our final in vitro activity assay in the presence of radioactively labeled alanyl-PG then revealed hydrolysis of the aminoacyl linkage, resulting in the formation of alanine and PG. Consequently, PA0919 was termed alanyl-PG hydrolase. The elucidated enzymatic activity implies a new regulatory circuit for the appropriate tuning of cellular alanyl-PG concentrations.

Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex.

Photosynthesis uses chlorophylls for the conversion of light into chemical energy, the driving force of life on Earth. During chlorophyll biosynthesis in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms, dark-operative protochlorophyllide oxidoreductase (DPOR), a nitrogenase-like metalloenzyme, catalyzes the chemically challenging two-electron reduction of the fully conjugated ring system of protochlorophyllide a. The reduction of the C-17=C-18 double bond results in the characteristic ring architecture of all chlorophylls, thereby altering the absorption properties of the molecule and providing the basis for light-capturing and energy-transduction processes of photosynthesis. We report the X-ray crystallographic structure of the substrate-bound, ADP-aluminium fluoride-stabilized (ADP·AlF(3)-stabilized) transition state complex between the DPOR components L(2) and (NB)(2) from the marine cyanobacterium Prochlorococcus marinus. Our analysis permits a thorough investigation of the dynamic interplay between L(2) and (NB)(2). Upon complex formation, substantial ATP-dependent conformational rearrangements of L(2) trigger the protein-protein interactions with (NB)(2) as well as the electron transduction via redox-active [4Fe-4S] clusters. We also present the identification of artificial "small-molecule substrates" of DPOR in correlation with those of nitrogenase. The catalytic differences and similarities between DPOR and nitrogenase have broad implications for the energy transduction mechanism of related multiprotein complexes that are involved in the reduction of chemically stable double and/or triple bonds.

Resistance phenotypes mediated by aminoacyl-phosphatidylglycerol synthases.

The specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine or with lysine catalyzed by aminoacyl-phosphatidylglycerol synthases (aaPGS) was shown to render various organisms less susceptible to antibacterial agents. This study makes use of Pseudomonas aeruginosa chimeric mutant strains producing lysyl-phosphatidylglycerol (L-PG) instead of the naturally occurring alanyl-phosphatidylglycerol (A-PG) to study the resulting impact on bacterial resistance. Consequences of such artificial phospholipid composition were studied in the presence of an overall of seven antimicrobials (ß-lactams, a lipopeptide antibiotic, cationic antimicrobial peptides [CAMPs]) to quantitatively assess the effect of A-PG substitution (with L-PG, L-PG and A-PG, increased A-PG levels). For the employed Gram-negative P. aeruginosa model system, an exclusive charge repulsion mechanism does not explain the attenuated antimicrobial susceptibility due to PG modification. Additionally, the specificity of nine orthologous aaPGS enzymes was experimentally determined. The newly characterized protein sequences allowed for the establishment of a significant group of A-PG synthase sequences which were bioinformatically compared to the related group of L-PG synthesizing enzymes. The analysis revealed a diverse origin for the evolution of A-PG and L-PG synthases, as the specificity of an individual enzyme is not reflected in terms of a characteristic sequence motif. This finding is relevant for future development of potential aaPGS inhibitors.

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).

Alanyl-phosphatidylglycerol synthase: mechanism of substrate recognition during tRNA-dependent lipid modification in Pseudomonas aeruginosa.

Bacterial lipid homeostasis plays an important role for the adaptation to changing environments and under conditions of antimicrobial treatment. The tRNA-dependent aminoacylation of the phospholipid phosphatidylglycerol catalysed by aminoacyl-phosphatidylglycerol synthases was shown to render various organisms less susceptible to antibacterial agents. Therefore, this type of enzyme might provide a new target to potentiate the efficacy of existing antimicrobials. This study makes use of the Pseudomonas aeruginosa alanyl-phosphatidylglycerol synthase to identify the minimal core domain of this transmembrane protein, which is capable of alanyl-phosphatidylglycerol biosynthesis. Using this catalytic fragment we established a reliable activity assay that was used to study the enzymatic mechanism by analysing an overall of 33 mutant proteins in vitro. Substrate recognition was analysed by using aminoacylated microhelices as analogues of the natural tRNA substrate. The enzyme even tolerated mutated versions of this minimal substrate, which indicates that neither the intact tRNA, nor the individual sequence of the acceptor stem is a determinant for substrate recognition. Furthermore, the analysis of derivatives of phosphatidylglycerol indicated that the polar head group of the phospholipid is specifically recognized by the enzyme, whereas modification of an individual fatty acid or even the deletion of a single fatty acid did not abolish A-PG synthesis.