Bioinformatic analysis of the fold type I PLP-dependent enzymes reveals determinants of reaction specificity in l-threonine aldolase from Aeromonas jandaei

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-5'-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.

Keywords: bioinformatics; enzyme catalysis; protein engineering; pyridoxal‐phosphate; threonine aldolase.

Figure 1

Aldol synthesis of l‐β‐hydroxy‐α‐amino acids catalysed by LTA.

Figure 2

Mechanism of catalysis by the enzymes from the aspartate aminotransferase superfamily.

Figure 3

Structure of LTA from Aeromonas jandaei (PDB code PDB:3WGB): (A) family‐specific (orange) and conserved (green) positions in the enzymes of aspartate aminotransferase superfamily are shown in subunit A of the homotetramer. (B) Interaction of PLP with family‐specific (orange) and conserved (green) positions in the active site.

Figure 4

Formation of the (A) internal aldimine and (B) external aldimine with the glycine substrate in the wild type LTAaj and its mutants at pH 8 (λ_max = 340 nm corresponds to gem‐diamine; λ_max = 390 nm – free PLP; λ_max = 410–420 nm – internal/external aldimine; λ_max = 495 nm – quinonoid complex).

Figure 5

The nomenclature of functional groups of PLP cofactor used in the manuscript.

Figure 6

Formation of the external aldimine via transimination reaction in the active site of the Fold type I PLP‐dependent enzymes.

Figure 7

Molecular dynamics snapshot of the LTA from Aeromonas jandaei with the l‐allo‐threonine substrate. The family‐specific residue His85 and His128 take turns in accommodating the OH‐group of the l‐allo‐threonine during the MD simulation (MD snapshots are taken at A: 15 ns; B: 40 ns).

Figure 8

PLP‐glycine Schiff base tautomers.

Figure 9

Coenzyme absorbance spectra obtained in the pH titration of the wild‐type LTAaj and its mutants. λ_max = 340 nm – enolimine; λ_max = 420 nm – ketoenamine. (A) wild type LTAaj; (B) R171F mutant of LTAaj; (C) C196T mutant of LTAaj.

Figure 10

(A) Transaminase activity in the mutants of LTA from Aeromonas jandaei with respect to wild type (100% corresponds to the transaminase activity in WT 0.6 U·mg⁻¹). (B) Racemization of l‐alanine to d‐alanine catalysed by R231A LTAaj (black line) and wild type LTAaj (dashed grey line). After 8 h: e.e.(WT) = 99%, e.e.(R231A) = 46%.

Figure 11

Proposed mechanism of l‐alanine racemization catalyzed by R231A LTA from Aeromonas jandaei.