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Zn-dependent Alkylsulfatases Hydrolysis of alkylsulfates is an important pathway for soil and other bacteria to mobilize sulfur. Three classes of sulfatases - divided according to their reaction mechanism - have been discovered, and the recently elucidated structure of SdsA1 from Pseudomonas aeruginosa is another example of the widely occurring family of metallo-ß-lactamases. This group of alkylsulfatases is characterized by two Zn 2+ atoms in the active site which activate a water molecule for nucleophilic attack on the sulfate group. SdsA1 mainly cleaves long-chain primary alkylsulfates (preferred substrate is dodecylsulfate) by stereoinversion. In other words, the hydroxyl group attacks the carbon atom in the course of the reaction. Recently, a novel enzyme could be identified in Pseudomonas DSM 6611 (termed PISA1 = Pseudomonas inverting alkylsulfatase 1) which mainly cleaves secondary alkylsulfates, for example 2octylsulfate exhibiting stereopreference for the (R)-stereoisomer. In contrast to the majority of hydrolases, which do not alter the stereochemistry of the substrate during catalysis, PISA1 is an attractive enzyme for the deracemisation of sec-alcohols. Analysis of the crystal structures of PISA1 and SdsA1 showed that the overall structure of both proteins is virtually identical and both enzymes largely share the same active-site architecture, such as a sulfate binding site (composed of two Arg), a nucleophile site composed of a binuclear Zn 2+ -cluster typical for metallo-ß-lactamases and an Asn/Thrhydrogen binding network for substrate positioning. However, the active site of PISA1 features several conspicuous amino acid exchanges (see figure below: in blue active side residues in SdsA1 and green those in PISA1). Phe/Gly Tyr/His Ala/Ile Tyr/Ser Met/Ser These amino acids are now subject of an extensive mutagenesis program to define their role in governing substrate preference of the reaction. This project is a close collaboration with Profs. Faber (biocatalysis) and Wagner (structure determination) from the University of Graz (thesis project of Tanja Knaus in our laboratory and Markus Schober in Prof. Faber’s laboratory). 12 Met/Ser Tyr/Ser Leu/Pro Ala/Ile
Doctoral thesis completed Alexandra Binter: Enzymes of nikkomycin biosynthesis Nikkomycins are peptide nucleosides which can be isolated from the fermentation broth of Streptomyces tendae and S. ansochromogenes. Due to their inhibition of chitin synthase they have fungicidal properties and a great potential as antibiotics. The peptide moiety is formed by hydroxypyridylhomothreonine, the nucleoside moiety consists of aminohexuronic acid with an N-glycosidically linked uracil or 4-formyl-4-imidazoline-2-one base. This work is intended to shed light onto the biosynthetic pathway that leads to the aminohexuronic acid moiety of nikkomycins. Commencing with the transfer of an enolpyruvyl moiety to the 3´position of UMP, which is catalyzed by NikO, a series of reactions leads to a rearrangement of the carbon skeleton and finally to the formation of aminohexuronic acid. The enzymes involved in these reactions are encoded on the nikIJKLMNO operon of S. tendae. The enzymes NikI, NikJ, NikK, NikL, NikM, and NikO were expressed heterologously in Escherichia coli, in order to characterize them biochemically and determine their role in the biosynthesis of nikkomycins. Furthermore, nikkomycin intermediates produced by Streptomyces mutants featuring disruptions of the respective genes should be analyzed, but no nikkomycins could be detected in their fermentation media. Comparisons of Nik enzyme sequences to those of known enzymes in the databases allow some predictions on the reactions each nik enzyme might catalyze. None of the enzymes was observed to convert 3´- EPUMP, the product of the NikO catalyzed reaction, so far. NikK is a pyridoxal-5-phosphate dependent aminotransferase, which is expected to introduce the amino group into the aminohexuronic acid moiety. Several amino acids were shown to serve as amino group donors, and L-glutamate was the most efficient one. NikJ possesses an iron-sulfur cluster and probably belongs to the family of radical SAM enzymes. Its role in nikkomycin biosynthesis is unclear, but it is expected to function as an oxidoreductase. Cleavage of Sadenosylmethionine could not be observed. Furthermore, NikS was expressed, an enzyme that is expected to play a role in the assembly of nikkomycins. International cooperations Maria Abramic, Ruder Boskovic Institute Zagreb, Croatia Steve Ealick, Cornell University, Ithaca, U.S.A. Toni Kutchan, Donald Danforth Plant Science Center, St. Louis, U.S.A. Shwu Liaw, National Yang-Ming University, Taipei, Taiwan Matthias Mack, Hochschule Mannheim, Germany Bruce Palfey, University of Michigan, Ann Arbor, U.S.A. Research projects FWF P24189: “Bacterial bioluminescence” FWF P22361: “Mechanism of redox controlled protein degradation” FWF P19858: “Enzymes of nikkomycin biosynthesis” FWF-Doktoratskolleg “Molecular Enzymology” WTZ Austria-Croatia “Structure-function relationships in metallopeptidases of the M49 family” 13
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