Thermostability and reactivity in organic solvent of O-phospho-l-serine sulfhydrylase from hyperthermophilic archaeon Aeropyrum pernix K1

Figures & data

Fig. 1. Schematic representation of the proposed reaction catalyzed by OPSS.

Fig. 2. Purification of His-OASS-B.

Notes: Samples at various stages during the purification procedure were subjected to SDS–PAGE (the result of 12.5% polyacrylamide gel). Lane 1, molecular weight marker; Lane 2, crude cellular lysate supernatant; Lane 3, the fraction of wash buffer; Lane 4, the fraction after the cell lysate supernatant was passed through the column; Lane 5, the fraction of elution buffer.

Table 1. Comparison of histidine-tagged OASS-B (His-OASS-B) with other reported OASS-B.

Reaction condition	Specific activity of other OASS-B (U/mg protein)	Specific activity of His-OASS-B (U/mg protein)	Relative activity
20 mM OAS,
2 mM Sodium sulfide
50 mM KPi pH 7.5	728	667 (21)^c	92
25 °C^a
10 mM OAS,
10 mM Sodium sulfide
100 mM KPi pH 7.0	150	150 (12)^c	100
37 °C^b

^a Ref.¹⁸⁾

^b Ref.¹²⁾

^c Values are expressed as the mean (standard deviation) of three experiments.

Fig. 3. Thermal stability of OPSS (squares) and His-OASS-B (circles).

Notes: The enzymes were incubated for 1 h at temperatures from 0 to 100 °C for OPSS, and from 0 to 70 °C for His-OASS-B, respectively. The relative remaining activities were determined from the activity without incubation as 100. Results are expressed as the mean ± standard deviation from three replicates.

Fig. 4. The reactivity of OPSS and His-OASS-B in the presence of polar and aprotic solvent, such as (A) N, N′-dimethylformamide (DMF) and (B) 1,4-dioxane.

Notes: Filled squares and circles show the relative remaining activity of OPSS and His-OASS-B, respectively. The enzymes were incubated for 3 min at temperatures at 80 °C for OPSS and 30 °C for His-OASS-B in the solution containing the aprotic solvent by 0–50% (v/v). The relative activities were determined from the activity without organic solvent as 100. Results are expressed as the mean ± standard deviation from three replicates.

Table 2. Electrostatic interaction in OPSS.

	Residues^a		Distance (Å)
1	ASP11.OD1	ARG41.NH2	2.43
2	ASP14.OD2	ARG140.NE	3.49
3	ARG35.NH1	GLU43.OE2	3.13
4	LYS48.NZ	GLU232.OE2	2.79
5	ARG57.NH1	ASP75.OD2	2.73
6	GLU69.OE2	ARG248.NH2	3.68
7	ASP89.OD2	ARG93.NH2	2.78
8	GLU92.OE1	ARG236.NH1	2.88
9	ARG111.NE	ASP362.OD1	2.87
10	LYS115.NZ	GLU241.OE1	2.81
11	GLU117.OE1	ARG129.NE	2.79
12	ARG129.NH2	GLU241.OE1	2.85
13	GLU133.OE2	ARG137.NH2	2.86
14	GLU133.OE1	ARG236.NE	2.79
15	ARG137.NH1	GLU232.OE1	2.68
16	ARG172.NH1	GLU217.OE2	2.75
17	ASP197.OD2	ARG209.NH1	4.10
18	ARG209.NE	ASP213.OD2	3.65
19	ARG254.NH2	ASP277.OD2	2.71
20	ARG254.NH1	GLU359.OE1	2.96
21	ARG281.NH2	ASP357.OD2	3.00
22	ARG298.NH1	GLU317.OE1	3.32
23	GLU322.OE2	LYS347.NZ	4.46
24	GLU322.OE2	LYS351.NZ	3.11
25	LYS352.NZ	ASP357.OD2	4.13
26	ASP369.OD1	LYS373.NZ	3.63

^a Atom names are according to the Protein Data Bank.

Table 3. Electrostatic interaction in OASS-B from E. coli.

	Residues^a		Distance (Å)
1	GLU5.OE2	LYS83.NZ	2.83
2	GLU26.OE1	ARG165.NH1	3.11
3	LYS30.NZ	GLU157.OE1	2.77
4	GLU32.OE1	ARG43.NE	2.75
5	ARG43.NH2	GLU157.OE1	2.88
6	GLU53.OE1	LYS83.NZ	3.84
7	ARG55.NH1	ASP145.OD2	2.9
8	GLU57.OE1	LYS136.NZ	3.08
9	ASP93.OD1	LYS115.NZ	3.21
10	ARG99.NH2	ASP281.OD2	4.38
11	ARG104.NH1	GLU109.OE1	3.27
12	ARG123.NE	ASP139.OD1	2.87
13	ARG187.NH1	ASP227.OD2	2.88
14	GLU202.OE1	ARG211.NE	3.09
15	ARG211.NH1	ASP231.OD2	3.07
16	ASP236.OD2	ARG265.NH1	4.34
17	GLU243.OE2	ARG247.NH1	2.82

^a Atom names are according to the Protein Data Bank.

Fig. 5. Intramolecular electrostatic interaction of (A) OPSS (orange, PDB code 3vsa, and chain A) and (B) OASS-B (cyan, PDB code 2v03) and (C) superposition of (A) and (B).

Notes: Red line shows electrostatic interaction formed between amino acid residues shown in Tables 2 and 3. Carbon, nitrogen, oxygen, and phosphorus atoms are shown as orange (OPSS) or cyan (OASS-B), blue, red, and pink balls, respectively. Illustration was created with MOE (ver.2011.10; Chemical Computing, Group, Montreal Canada).

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