04 Fakultät Energie-, Verfahrens- und Biotechnik
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Item Open Access Microbial aldolases as C-C bonding enzymes : investigation of structural-functional characteristics and application for streoselective reactions(2006) Inoue, Tomoyuki; Sprenger, Georg (Prof. Dr.)Carbon-carbon bonding enzymes can be attractive alternatives to standard chemical methods by allowing chiral control, taking advantage of mild reaction conditions and minimizing the use of protecting groups in reactions. Fructose-6-phosphate aldolase (FSA) from Escherichia coli catalyses reversibly the cleavage of D-fructose-6-phosphate into dihydroxyacetone (DHA) and D-glyceraldehyde-3-phosphate (GAP). In addition, the enzyme creates new building blocks with 3S, 4R configuration by use of DHA as donor and various aldehydes as acceptor. The goal of this work was to investigate FSA and compare it with the transaldolase from Bacillus subtilis (TALBsu) which is similar both in sequence and structure to FSA (30% identical in amino acid residues). This should help to elucidate relationships between structure and function of both enzymes, and possible applications for FSA and TALBsu for the production of valuable sugars or sugar derivatives. Both fsa and talBsu genes were cloned with Histidine tags (6x or 10x His-tag) at the N- or C-termini to facilitate purification of the proteins. However, none of these fusion proteins (N-tagged FSA, C-tagged FSA, N-tagged TALBsu, C-tagged TALBsu) retained the complete enzyme activity. Both N-tagged FSA and TALBsu did not bind to Ni-NTA column, whereas both C-tagged enzymes bound to the resin and they could be purified. Concerning quaternary structures, N- or C-tagged TALBsu formed dimer or pentamer, while both His-tagged FSAs kept the decamer structures. This result demonstrated that His-tag would give negative influences on both FSA and TALBsu, and it was unsuitable for assay of those enzymes. In addition, FSA had different structural stability from that of TALBsu. To examine the importance of several residues at the active center in FSA, three FSA mutants (Q59E, Y131A and Y131F) were prepared so as to have the corresponding amino acid residues that are present in TALBsu and several other TALs (Gln -> Glu, Tyr -> Phe). The purified FSA Q59E protein retained approximately 66% of the wild type (WT) activity, whereas both Y131A and Y131F were completely inactive, though all three retained their decameric structures. From three-dimensional structural analysis of FSA Q59E (by the group of G. Schneider at Karolinska Institute, Stockholm), it appeared that the mutant protein's structure is identical to WT. Each mutant lost stability at high temperature and was thus denatured by heat treatment (75°C, 40min). This suggested that the Tyr131 residue has an important role for FSA activity. Indeed, a hydroxyl group at the phenyl moiety of the residue appears to be indispensable for the catalytic reaction, as the structural balance of FSA was lost by the alteration of Tyr131. Another mutant of FSA, A129S, was assayed for its synthetic capability and compared with FSA WT. Both WT and A129S catalyzed two reactions using DHA as donor with formaldehyde or glycolaldehyde as acceptor and produced S-erythrulose or D-xylulose, respectively. However, A129S showed much higher activity thus yielding larger amounts of products. On the other hand, almost no difference was observed in catalytic ability between WT and A129S for a reaction using hydroxyacetone as donor. It is supposed that a hydroxyl moiety of DHA interacts with the hydroxyl group of Ser129 in FSA A129S via a hydrogen bond and changes the affinity of the enzyme towards DHA. As further synthetic reactions, aminoaldehydes were assayed as acceptors for FSA. FSA recognized N-Cbz-3-aminopropanal with DHA and an aldol adduct (a precursor of fagomine) was produced even at 4 °C (performed by the group of P. Clapes in CSIC, Barcelona). This indicates a high utility of FSA in organic syntheses, the enzyme indeed could recognize large molecules including a benzene ring as acceptor and retains catalytic ability at rather low temperature. To evolve diverse catalytic abilities of FSA and progress further application of the enzyme, random mutagenesis was adopted by use of error prone PCR technique. As a result, various mutant proteins were acquired with the alteration of 1 to 6 residues per gene. This implies that FSA mutants can be prepared with this technique and enzyme libraries could be created. To compare FSA with TALBsu in structure, wild-type TALBsu was cloned in E. coli and purified. Three-dimensional structure analysis (by T. Sandalova in the group of G. Schneider in Karolinska Institute, Stockholm) revealed that the enzyme is a decameric protein (10 subunits of 23kDa) resulting from the dimerization of two identical pentamers. This makes TALBsu highly similar to FSA in structure with the exception only of a shorter C-terminal helix. TALBsu was tolerant of high temperature as FSA (at 75°C for 30-40min), and recognized different aldehydes or their phosphate derivatives as acceptors (Fru6P as donor). DHA was utilized as donor at a specific activity of about 10% of a reaction using Fru6P. Eight chimera proteins (chimera1-8) consisting of parts of FSA and progressively truncated TALBsu were designed and overexpressed in E. coli to probe structural determinants for each enzyme. However, several chimera samples (chimera1, 3, 6) showed only faint protein bands on SDS-PAGE and two (chimera2, 7) were not detected. All cell-free extracts of chimera proteins showed neither FSA nor TALBsu activity.Item Open Access Polyphosphat : ein unterschätztes Molekül(2022) Jendrossek, Dieter; Hildenbrand, Jennie C.Polyphosphate (polyP) is an inorganic biopolymer ubiquitously present in all species. It has a variety of functions ranging from that of a reservoir for phosphorous in many microorganisms to functions in blood coagulation and plays a role in neurogenerative diseases in humans. Here, we provide a summary of the structure and functions that have been addressed to polyP in microorganisms.Item Open Access Enzyme, Gene und Mechanismen des oberen Abbauweges von Pikrinsäure und 2,4-Dinitrophenol durch Nocardioides simplex FJ2-1A(2001) Ebert, Sybille; Knackmuss, Hans-Joachim (Prof. Dr.)Der Bakterienstamm Nocardioides simplex FJ2-1A baut 2,4-Dinitrophenol (2,4-DNP) und Pikrinsäure unter aeroben Bedingungen vollständig ab. Hydrid wird auf den aromatischen Ring des Nitroaromaten übertragen. Dabei entsteht der jeweilige Hydrid-Meisenheimer-Komplex als Produkt. Diese initiale Reduktion wird von einem Enzymsystem katalysiert. Koenzym F420, welches als methanogener Kofaktor bekannt ist, wurde als Mediator der Hydridübertragung identifiziert. Von NADPH wird Hydrid mittels einer NADPH-abhängigen F420-Reduktase auf Koenzym F420 übertragen. Der sich anschließende Hydridtransfer auf das aromatische System des Nitroaromaten wird durch eine Hydridtransferase katalysiert. Die N-terminalen Sequenzen der Proteine dienten zur Ableitung von Oligonukleotiden für die Herstellung einer Sonde mittels PCR. Ein 7.2 kb großes DNA Fragment, das die Gene der beiden Enzyme beinhaltet, wurde isoliert und sequenziert. Bei Datenbankvergleichen zeigt die F420-Reduktase Ähnlichkeiten zu F420-abhängigen NADP+-Reduktasen aus Archaea und Streptomyceten. Die Hydridtransferase ist N5,N10-Methylen-tetrahydromethanopterin-Reduktasen ähnlich. Der Dihydrid-Komplex von Pikrat wurde als Produkt einer zweifachen Hydridübertragung auf das aromatische Ringsystem von Pikrat durch das Enzymsystem identifiziert. Der Dihydrid Komplex des Pikrats wurde 1H- und 13C-NMR spektroskopisch untersucht. Er wird enzymatisch unter Elimination von Nitrit und der Bildung des Hydrid-Meisenheimer-Komplexes von 2,4-DNP umgesetzt. Die nitriteliminierende Aktivität wurde mit FPLC angereichert. Als Produkt der Reduktion von 2,4-DNP durch das Hydrid übertragende Enzymsystem entsteht der korrespondierende Hydrid-Meisenheimer-Komplex. Als Folge dieser Untersuchungen ergibt sich für Nocardioides simplex FJ2-1A ein konvergenter Abbauweg von Pikrat und 2,4-DNP.