K30, H150, and H168 are essential residues for coordinating pyridoxal 5'-phosphate of O-acetylserine sulfhydrylase from Acidithiobacillus ferrooxidans

Abstract

O-acetylserine sulfhydrylase (OASS) is a key enzyme involved in the pathway of the cysteine biosynthesis. The gene of OASS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and expressed in E. coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. Colors and UV-vis scanning results of the recombinant protein confirmed that it was a pyridoxal 5'-phosphate (PLP)-containing protein. Sequence alignment and site-directed mutation of the enzyme revealed that the cofactor PLP is covalently bound in Schiff base linkage with K30, as well as the two residues H150 and H168 were the crucial residues for PLP binding and stabilization.

Fig. 1

Coomassie blued-stained SDS–PAGE of the purified OASS from A. ferrooxidans and its mutant proteins. Lane 1 molecular mass standards, lane 2 purified OASS wild-type, lane 3 purified OASS mutant K30A, lane 4 purified OASS mutant K41A, lane 5 purified OASS mutant H150A, lane 6 purified OASS mutant H168A

Fig. 2

UV–vis scanning of apo and holocysM from A. ferrooxidans. Solid line indicates the holocysM, dashed line indicates apocysM without the PLP cofactor

Fig. 3

Emission spectrum upon excitation at 298 nm and 412 nm (slitex = 5 nm, slitem = 5 nm) of OASS from A. ferrooxidans

Fig. 4

The sequence alignment of OASS from A. ferrooxidans and other sources. A. ferrooxidans from A. ferrooxidans ATCC 23270; H. arsenicoxydans from Herminiimonas arsenicoxydans; A. vinosum from Allochromatium vinosum; H. halophila SL1 from Halorhodospira halophila SL1; B. cepacia from Burkholderia cepacia. The four residues proposed to coordinate a PLP cofactor are marked in asterisk. The highly conserved residues are marked in black

Fig. 5

UV–vis scanning of K30, H150, H168, and K41 OASS mutant enzymes