Abstract
Understanding the role of specific amino acid residues in the molecular
mechanism of a protein’s function is one of the most challenging problems
in modern biology. A systematic bioinformatic analysis of protein families
and superfamilies can help in the study of structure–function relationships
and in the design of improved variants of enzymes/proteins, but represents a
methodological challenge. The pyridoxal-50-phosphate (PLP)-dependent
enzymes are catalytically diverse and include the aspartate aminotransferase
superfamily which implements a common structural framework known as
type fold I. In this work, the recently developed bioinformatic online methods
Mustguseal and Zebra were used to collect and study a large representative
set of the aspartate aminotransferase superfamily with high structural,
but low sequence similarity to L-threonine aldolase from Aeromonas jandaei
(LTAaj), in order to identify conserved positions that provide general properties
in the superfamily, and to reveal family-specific positions (FSPs)
responsible for functional diversity. The roles of the identified residues in
the catalytic mechanism and reaction specificity of LTAaj were then studied
by experimental site-directed mutagenesis and molecular modelling. It was
shown that FSPs determine reaction specificity by coordinating the PLP
cofactor in the enzyme’s active centre, thus influencing its activation and the
tautomeric equilibrium of the intermediates, which can be used as hotspots
to modulate the protein’s functional properties. Mutagenesis at the selected
FSPs in LTAaj led to a reduction in a native catalytic activity and increased
the rate of promiscuous reactions. The results provide insight into the structural
basis of catalytic promiscuity of the PLP-dependent enzymes and
demonstrate the potential of bioinformatic analysis in studying structure–
function relationship in protein superfamilies.
mechanism of a protein’s function is one of the most challenging problems
in modern biology. A systematic bioinformatic analysis of protein families
and superfamilies can help in the study of structure–function relationships
and in the design of improved variants of enzymes/proteins, but represents a
methodological challenge. The pyridoxal-50-phosphate (PLP)-dependent
enzymes are catalytically diverse and include the aspartate aminotransferase
superfamily which implements a common structural framework known as
type fold I. In this work, the recently developed bioinformatic online methods
Mustguseal and Zebra were used to collect and study a large representative
set of the aspartate aminotransferase superfamily with high structural,
but low sequence similarity to L-threonine aldolase from Aeromonas jandaei
(LTAaj), in order to identify conserved positions that provide general properties
in the superfamily, and to reveal family-specific positions (FSPs)
responsible for functional diversity. The roles of the identified residues in
the catalytic mechanism and reaction specificity of LTAaj were then studied
by experimental site-directed mutagenesis and molecular modelling. It was
shown that FSPs determine reaction specificity by coordinating the PLP
cofactor in the enzyme’s active centre, thus influencing its activation and the
tautomeric equilibrium of the intermediates, which can be used as hotspots
to modulate the protein’s functional properties. Mutagenesis at the selected
FSPs in LTAaj led to a reduction in a native catalytic activity and increased
the rate of promiscuous reactions. The results provide insight into the structural
basis of catalytic promiscuity of the PLP-dependent enzymes and
demonstrate the potential of bioinformatic analysis in studying structure–
function relationship in protein superfamilies.
| Originalsprache | englisch |
|---|---|
| Seitenumfang | 16 |
| Fachzeitschrift | FEBS Open Bio |
| DOIs | |
| Publikationsstatus | Elektronische Veröffentlichung vor Drucklegung. - 8 Mai 2018 |
Fields of Expertise
- Human- & Biotechnology