Jürgen Moser

Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes.

‘Radical SAM’ enzymes generate catalytic radicals by combining a 4Fe–4S cluster and S‐adenosylmethionine (SAM) in close proximity. We present the first crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen‐independent coproporphyrinogen III oxidase, at 2.07 Å resolution. HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe–4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 Å) and a neighboring sulfur of the cluster (3.6 Å), allowing single electron transfer from the 4Fe–4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5′‐deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the first. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme.

V-Shaped Structure of Glutamyl-tRNA Reductase, the First Enzyme of tRNA-Dependent Tetrapyrrole Biosynthesis.

Processes vital to life such as respiration and photosynthesis critically depend on the availability of tetrapyrroles including hemes and chlorophylls. tRNA‐dependent catalysis generally is associated with protein biosynthesis. An exception is the reduction of glutamyl‐tRNA to glutamate‐1‐semialdehyde by the enzyme glutamyl‐tRNA reductase. This reaction is the indispensable initiating step of tetrapyrrole biosynthesis in plants and most prokaryotes. The crystal structure of glutamyl‐tRNA reductase from the archaeon Methanopyrus kandleri in complex with the substrate‐like inhibitor glutamycin at 1.9 Å resolution reveals an extended yet planar V‐shaped dimer. The well defined interactions of the inhibitor with the active site support a thioester‐mediated reduction process. Modeling the glutamyl‐tRNA onto each monomer reveals an extensive protein–tRNA interface. We furthermore propose a model whereby the large void of glutamyl‐tRNA reductase is occupied by glutamate‐1‐semialdehyde‐1,2‐mutase, the subsequent enzyme of this pathway, allowing for the efficient synthesis of 5‐aminolevulinic acid, the common precursor of all tetrapyrroles.

Bacterial heme biosynthesis and its biotechnological application

Proteins carrying a prosthetic heme group are vital parts of bacterial energy conserving and stress response systems. They also mediate complex enzymatic reactions and regulatory processes. Here, we review the multistep biosynthetic pathway of heme formation including the enzymes involved and reaction mechanisms. Potential biotechnological implications are discussed.

Adaptation of Pseudomonas aeruginosa to various conditions includes tRNA-dependent formation of alanyl-phosphatidylglycerol

The opportunistic bacterium Pseudomonas aeruginosa synthesizes significant amounts of an additional phospholipid, identified as 2′ alanyl‐phosphatidylglycerol (A‐PG), when exposed to acidic growth conditions. At pH 5.3 A‐PG contributed up to 6% to the overall lipid content of the bacterium. Sequence analysis of P. aeruginosa revealed open reading frame PA0920 showing 34% sequence identity to a protein from Staphylococcus aureus involved in tRNA‐dependent formation of lysyl‐phosphatidylglycerol. The P. aeruginosa deletion mutant ΔPA0920 failed to synthesize A‐PG. Heterologous overproduction of PA0920 in Escherichia coli resulted in the formation of significant amounts of A‐PG, otherwise not synthesized by E. coli. Consequently, the protein encoded by PA0920 was named A‐PG synthase. The enzyme was identified as an integral component of the inner membrane. The protein was partially purified by detergent solubilization and subjected to an in vitro activity assay. tRNAAla‐dependent catalysis was demonstrated. Transcriptional analysis of the corresponding gene in P. aeruginosa using lacZ reporter gene fusion under various pH conditions indicated a 4.4‐fold acid‐activated transcription. A phenotype microarray analysis was used to identify further conditions for A‐PG function.

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 Km values of 6.1 μm for protochlorophyllide, 13.5 μm for ATP, and 52.7 μm 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 (Cys97 and Cys131) of homodimeric BchL2 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, Lys10 and Leu126 are crucial to ATP-driven electron transfer from BchL2. 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 (Cys21, Cys46, and Cys103) and one from BchB (Cys94), 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.

Escherichia coli Glutamyl-tRNA Reductase TRAPPING THE THIOESTER INTERMEDIATE

In the first step of tetrapyrrole biosynthesis inEscherichia coli, glutamyl-tRNA reductase (GluTR, encoded by hemA) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde. Soluble homodimeric E. coli GluTR was made by co-expressing thehemA gene and the chaperone genes dnaJK andgrpE. During Mg2+-stimulated catalysis, the reactive sulfhydryl group of Cys-50 in the E. coli enzyme attacks the α-carbonyl group of the tRNA-bound glutamate. The resulting thioester intermediate was trapped and detected by autoradiography. In the presence of NADPH, the end product, glutamate-1-semialdehyde, is formed. In the absence of NADPH, E. coli GluTR exhibited substrate esterase activity. The in vitro synthesized unmodified glutamyl-tRNA was an acceptable substrate for E. coli GluTR. Eight 5-aminolevulinic acid auxotrophic E. coli hemA mutants were genetically selected, and the corresponding mutations were determined. Most of the recombinant purified mutant GluTR enzymes lacked detectable activity. Based on the Methanopyrus kandleri GluTR structure, the positions of the amino acid exchanges are close to the catalytic domain (G7D, E114K, R314C, S22L/S164F, G44C/S105N/A326T, G106N, S145F). Only GluTR G191D (affected in NADPH binding) revealed esterase but no reductase activity.

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·AlF3-stabilized) transition state complex between the DPOR components L2 and (NB)2 from the marine cyanobacterium Prochlorococcus marinus. Our analysis permits a thorough investigation of the dynamic interplay between L2 and (NB)2. Upon complex formation, substantial ATP-dependent conformational rearrangements of L2 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.

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.

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 (β-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.

Large scale production of biologically active Escherichia coli glutamyl-tRNA reductase from inclusion bodies

Glutamyl-tRNA reductase catalyzes the initial step of tetrapyrrole biosynthesis in plants and prokaryotes. Recombinant Escherichia coli glutamyl-tRNA reductase was purified to apparent homogeneity from an overproducing E. coli strain by a two-step procedure yielding 5.6 mg of enzyme per gram of wet cells with a specific activity of 0.47 micromol min(-1)mg(-1). After recombinant production, denatured glutamyl-tRNA reductase from inclusion bodies was renatured by an on-column refolding procedure. Residual protein aggregates were removed using Superdex 200 gel-filtration chromatography. Solubility, specific activity, and long-term storage properties were improved compared to previous protocols. Obtained enzyme amounts of high purity now allow the research on the recognition mechanism of tRNAGlu and high-throughput inhibitor screening.