Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

Gjalt G Wybenga,Ciprian G Crismaru,Dick B Janssen,Bauke W Dijkstra

doi:10.1074/jbc.m112.375238

Abstract

Chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure β-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-β-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a β-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A β-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both β- and α-amino acids.

Highlights

␤-Amino acids occur as precursors of many natural and synthetic compounds that display a wide range of pharmacological activities
In the second half-reaction, the amino group of pyridoxamine phosphate (PMP) is transferred to a keto acid, which generates a new amino compound (Fig. 1) [6]
The production of ␤-amino acids using Pyridoxal 5Ј-phosphate (PLP)-dependent aminotransferases has been demonstrated for an ␻-transaminase from Polaromonas sp. strain JS666 [7] and a ␤-transaminase from Mesorhizobium sp. strain LUK (MesAT) [8]

Summary

Introduction

␤-Amino acids occur as precursors of many natural and synthetic compounds that display a wide range of pharmacological activities. The overall structure is similar to that of aspartate aminotransferase, the archetypical representative of fold type I aminotransferases (Z-score of 21, r.m.s.d. of 4.2 Å for 309 C␣ atoms, and 16% sequence identity; Protein Data Bank code 1BKG) [29, 30], and AroAT from P. denitrificans (Z-score of 21, r.m.s.d. of 4.8 Å for 326 C␣ atoms, and 15% sequence identity; code 1AY4) [11, 31], which is specific for L-␣-Phe. PLP Cofactor—Each MesAT monomer contains a PLP cofactor covalently anchored via an imine bond (Schiff base) to the ⑀-amino group of Lys-280.

Results

Conclusion