Structural and Mutagenesis Studies of the Thiamine-Dependent, Ketone-Accepting YerE from Pseudomonas protegens.

A wide range of thiamine diphosphate (ThDP)-dependent enzymes catalyze the benzoin-type carboligation of pyruvate with aldehydes. A few ThDP-dependent enzymes, such as YerE from Yersinia pseudotuberculosis (YpYerE), are known to accept ketones as acceptor substrates. Catalysis by YpYerE gives access to chiral tertiary alcohols, a group of products difficult to obtain in an enantioenriched form by other means. Hence, knowledge of the three-dimensional structure of the enzyme is crucial to identify structure-activity relationships. However, YpYerE has yet to be crystallized, despite several attempts. Herein, we show that a homologue of YpYerE, namely, PpYerE from Pseudomonas protegens (59 % amino acid identity), displays similar catalytic activity: benzaldehyde and its derivatives as well as ketones are converted into chiral 2-hydroxy ketones by using pyruvate as a donor. To enable comparison of aldehyde- and ketone-accepting enzymes and to guide site-directed mutagenesis studies, PpYerE was crystallized and its structure was determined to a resolution of 1.55 Å.

Engineered Bacillus pumilus laccase-like multi-copper oxidase for enhanced oxidation of the lignin model compound guaiacol.

Laccases and laccase-like multi-copper oxidases (LMCOs) are versatile and robust biocatalysts applied in a variety of oxidative processes, and various studies have attempted to improve their catalytic activity. Here we report the engineering of a bacterial LMCO for enhanced oxidation of the lignin-related compound guaiacol by a combination of structure-guided mutagenesis and DNA shuffling. Mutant L9 showed a 1.39 mM Km for guaiacol and a 2.5-fold increase in turnover rate (kcat/Km = 2.85·104 M-1s-1).

Increased efficiency of Campylobacter jejuni N-oligosaccharyltransferase PglB by structure-guided engineering.

Conjugate vaccines belong to the most efficient preventive measures against life-threatening bacterial infections. Functional expression of N-oligosaccharyltransferase (N-OST) PglB of Campylobacter jejuni in Escherichia coli enables a simplified production of glycoconjugate vaccines in prokaryotic cells. Polysaccharide antigens of pathogenic bacteria can be covalently coupled to immunogenic acceptor proteins bearing engineered glycosylation sites. Transfer efficiency of PglBCj is low for certain heterologous polysaccharide substrates. In this study, we increased glycosylation rates for Salmonella enterica sv. Typhimurium LT2 O antigen (which lacks N-acetyl sugars) and Staphylococcus aureus CP5 polysaccharides by structure-guided engineering of PglB. A three-dimensional homology model of membrane-associated PglBCj, docked to the natural C. jejuni N-glycan attached to the acceptor peptide, was used to identify potential sugar-interacting residues as targets for mutagenesis. Saturation mutagenesis of an active site residue yielded the enhancing mutation N311V, which facilitated fivefold to 11-fold increased in vivo glycosylation rates as determined by glycoprotein-specific ELISA. Further rounds of in vitro evolution led to a triple mutant S80R-Q287P-N311V enabling a yield improvement of S. enterica LT2 glycoconjugates by a factor of 16. Our results demonstrate that bacterial N-OST can be tailored to specific polysaccharide substrates by structure-guided protein engineering.

Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB.

BACKGROUND: Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. RESULTS: In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette saturation mutagenesis. With the exception of I571C only hydrophobic residues were found in active variants. Variant I571V performed equally well as the wild type, cysteine at the same position reduced glycoprotein yield slightly, while a change to phenylalanine reduced activity by a factor of three. CONCLUSIONS: This study provides novel structure-function relationships for the periplasmic domain of the Campylobacter jejuni N-oligosaccharyltransferase PglB and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered E. coli system.

A simple HPLC-MS method for the quantitative determination of the composition of bacterial medium chain-length polyhydroxyalkanoates.

Bacterial poly(hydroxyalkanoates) (PHAs) vary in the composition of their monomeric units. Besides saturated side-chains, unsaturated ones can also be found. The latter leads to unwanted by-products (THF ester, secondary alcohols) during acidic cleavage of the polymer backbone in the conventional analytical assays. To prevent these problems, we developed a new method for the reductive depolymerization of medium chain-length PHAs, leading to monomeric diols that can be separated and quantified by HPLC/MS. Reduction is performed at room temperature with lithium aluminum hydride within 5-15 min. The new method is faster and simpler than the previous ones and is quantitative. The results are consistent with the ones obtained by quantitative (1)H NMR.