Project Details
Projekt
Investigation of the structure function relationships within the superfamilies of PLP-dependent transaminases
Subject Area
Biochemistry
Term
from 2015 to 2020
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 277430376
In the era of genome sequencing the precise prediction of the physiological function of the encoded proteins is an important prerequisite to decipher the metabolic performance of an organism. Despite modern bioinformatic methods, usually only the reaction specificity (i.e. class III transaminase) can be predicted. The substrate specificity however can only be predicted in a limited fashion. To allow for a reliable annotation a detailed understanding of the structure-function relationships is required as well as the ability to derive these factors at the level of protein sequences.PLP-dependent enzymes catalyze an impressive array of reactions. In the superfamilies of the PLP fold classes I and IV we recently identified in our previous work a platform of 20 (R)-selective amine transaminases (ATA). Recently, we succeeded in the determination of the crystal structure of an (R)-ATA and could elucidate the function of four previously uncharacterized PLP-dependent enzymes of which the structures were already deposited in the pdb-protein database. On the basis of these investigations it is the aim of this research project to investigate the structure-function relationships of the enzymes belonging to the subfamily class III and to the PLP fold class IV transaminases. Preliminary sequence analyses revealed that the substrate specificity of >12 different enzyme activities within the class III transaminases correlate with amino acid sequence patterns (by focussing on the active site). By means of a robot-assisted biochemical characterizations of a multitude of further proteins we will (i) identify and experimentally confirm the factors, which according to our hypothesis, determine the substrate specificity, (ii) discover further so far unknown factors and (iii) identify novel yet unexplored enzyme activities in these subfamilies. By transfer of a valine transaminase into a (R)-ATA and vice versa through experimental mutagenesis studies we will gain a much deeper understanding of the structure-function relationship in the PLP fold class IV. In order to understand how substrate binding as well as the flexibility of the protein determines the substrate specifity of the protein during catalysis, we will predict via computer simulations mutations and characterize the corresponding enzyme variants experimentally. Altogether we expect a significanlty better understanding of the molecular reasons for the reaction and substrate specificity, which are very useful for a more precise annotation of PLP-dependent transaminases.
DFG Programme
Research Grants
