NDLI: Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases

Content Provider	Springer Nature Link
Author	Knight, Anders M. bili, Alberto Bergh, Tom Genz, Maika Joosten, Henk Jan Albrecht, Dirk Riedel, Katharina Pavlidis, Ioannis V. Bornscheuer, Uwe T.
Copyright Year	2016
Abstract	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.
Starting Page	1499
Ending Page	1507
Page Count	9
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	101
Issue Number	4
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2016-10-27
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Decarboxylase PLP-dependent enzymes Protein-function analysis Racemase Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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