Alberto Nobili
University of Greifswald
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Featured researches published by Alberto Nobili.
Biotechnology Advances | 2015
Fabian Steffen-Munsberg; Clare Vickers; Hannes Kohls; Henrik Land; Hendrik Mallin; Alberto Nobili; Lilly Skalden; Tom van den Bergh; Henk-Jan Joosten; Per Berglund; Matthias Höhne; Uwe T. Bornscheuer
In this review we analyse structure/sequence-function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence-function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof.
Chemcatchem | 2015
Alberto Nobili; Fabian Steffen-Munsberg; Hannes Kohls; Ivan Trentin; Carola Schulzke; Matthias Höhne; Uwe T. Bornscheuer
Although the amine transaminase from Vibrio fluvialis has often been applied as a catalyst for the biocatalytic preparation of various chiral primary amines, it is not suitable for the transamination of α‐hydroxy ketones and aryl‐alkyl ketones bearing an alkyl substituent larger than a methyl group. We addressed this problem through a systematic mutagenesis study of active site residues to expand its substrate scope towards two bulky ketones. We identified two mutants (F85L/V153A and Y150F/V153A) showing 30‐fold increased activity in the conversion of (S)‐phenylbutylamine and (R)‐phenylglycinol, respectively. Notably, they facilitated asymmetric synthesis of these amines with excellent enantiomeric purities of 98 % ee.
FEBS Journal | 2013
Alberto Nobili; Markus Gall; Ioannis V. Pavlidis; Mark L. Thompson; Marlen Schmidt; Uwe T. Bornscheuer
Two libraries of simultaneous double mutations in the active site region of an esterase from Bacillus stearothermophilus were constructed to improve the enantioselectivity in the hydrolysis of tetrahydrofuran‐3‐yl acetate. As screening of large mutant libraries is hampered by the necessity for GC/MS analysis, mutant libraries were designed according to a ‘small but smart’ concept. The design of focused libraries was based on data derived from a structural alignment of 3317 amino acid sequences of α/β‐hydrolase fold enzymes with the bioinformatic tool 3dm. In this way, the number of mutants to be screened was substantially reduced as compared with a standard site‐saturation mutagenesis approach. Whereas the wild‐type esterase showed only poor enantioselectivity (E = 4.3) in the hydrolysis of (S)‐tetrahydrofuran‐3‐yl acetate, the best variants obtained with this approach showed increased E‐values of up to 10.4. Furthermore, some variants with inverted enantiopreference were found.
ChemBioChem | 2015
Lilly Skalden; Christin Peters; Jonathan Dickerhoff; Alberto Nobili; Henk-Jan Joosten; Klaus Weisz; Matthias Höhne; Uwe T. Bornscheuer
Amine transaminases (ATAs) are powerful enzymes for the stereospecific production of chiral amines. However, the synthesis of amines incorporating more than one stereocenter is still a challenge. We developed a cascade synthesis to access optically active 3‐alkyl‐substituted chiral amines by combining two asymmetric synthesis steps catalyzed by an enoate reductase and ATAs. The ATA wild type from Vibrio fluvialis showed only modest enantioselectivity (14 % de) in the amination of (S)‐3‐methylcyclohexanone, the product of the enoate‐reductase‐catalyzed reaction step. However, by protein engineering we created two variants with substantially improved diastereoselectivities: variant Leu56Val exhibited a higher R selectivity (66 % de) whereas the Leu56Ile substitution caused a switch in enantiopreference to furnish the S‐configured diastereomer (70 % de). Addition of 30 % DMSO further improved the selectivity and facilitated the synthesis of (1R,3S)‐1‐amino‐3‐methylcyclohexane with 89 % de at 87 % conversion.
ChemBioChem | 2015
Alberto Nobili; Yifeng Tao; Ioannis V. Pavlidis; Tom van den Bergh; Henk-Jan Joosten; Tianwei Tan; Uwe T. Bornscheuer
In order to improve the efficiency of directed evolution experiments, in silico multiple‐substrate clustering was combined with an analysis of the variability of natural enzymes within a protein superfamily. This was applied to a Pseudomonas fluorescens esterase (PFE I) targeting the enantioselective hydrolysis of 3‐phenylbutyric acid esters. Data reported in the literature for nine substrates were used for the clustering meta‐analysis of the docking conformations in wild‐type PFE I, and this highlighted a tryptophan residue (W28) as an interesting target. Exploration of the most frequently, naturally occurring amino acids at this position suggested that the reduced flexibility observed in the case of the W28F variant leads to enhancement of the enantioselectivity. This mutant was subsequently combined with mutations identified in a library based on analysis of a correlated mutation network. By interrogation of <80 variants a mutant with 15‐fold improved enantioselectivity was found.
PLOS ONE | 2017
Tom van den Bergh; Giorgio Tamo; Alberto Nobili; Yifeng Tao; Tianwei Tan; Uwe T. Bornscheuer; Remko Kuipers; Bas Vroling; René M. de Jong; Kalyanasundaram Subramanian; Peter J. Schaap; Tom Desmet; Bernd Nidetzky; Gert Vriend; Henk-Jan Joosten
CorNet is a web-based tool for the analysis of co-evolving residue positions in protein super-family sequence alignments. CorNet projects external information such as mutation data extracted from literature on interactively displayed groups of co-evolving residue positions to shed light on the functions associated with these groups and the residues in them. We used CorNet to analyse six enzyme super-families and found that groups of strongly co-evolving residues tend to consist of residues involved in a same function such as activity, specificity, co-factor binding, or enantioselectivity. This finding allows to assign a function to residues for which no data is available yet in the literature. A mutant library was designed to mutate residues observed in a group of co-evolving residues predicted to be involved in enantioselectivity, but for which no literature data is available yet. The resulting set of mutations indeed showed many instances of increased enantioselectivity.
Applied Microbiology and Biotechnology | 2017
Anders M. Knight; Alberto Nobili; Tom van den Bergh; Maika Genz; Henk-Jan Joosten; Dirk Albrecht; Katharina Riedel; Ioannis V. Pavlidis; Uwe T. Bornscheuer
Pyridoxal-5′-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form.
Applied Microbiology and Biotechnology | 2014
Markus Gall; Alberto Nobili; Ioannis V. Pavlidis; Uwe T. Bornscheuer
Protein Engineering Design & Selection | 2017
Andreas Schwarte; Maika Genz; Lilly Skalden; Alberto Nobili; Clare Vickers; Okke Melse; Remko Kuipers; Henk-Jan Joosten; Jan Štourač; Jaroslav Bendl; Jon Black; Peter Haase; Coos Baakman; Jiri Damborsky; Uwe T. Bornscheuer; Gert Vriend; Hanka Venselaar
Neuro-oncology | 2018
Karen Wright; Alberto Nobili; Steven C Neier; Pratiti Bandopadhayay; Colby P Ledoux; Susanna Baumeister; Hye-Jung Kim; Carl D. Novina; Mark W. Kieran