Limits...
Cloning, expression, purification, and characterization of glutaredoxin from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178.

Wang Q, Hou Y, Shi Y, Han X, Chen Q, Hu Z, Liu Y, Li Y - Biomed Res Int (2014)

Bottom Line: Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide.Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC.It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl.

View Article: PubMed Central - PubMed

Affiliation: School of Marine and Technology, Harbin Institute of Technology, Weihai 264209, China.

ABSTRACT
Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx) with 270 nucleotides was isolated from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. Recombinant PsGrx (rPsGrx) stably expressed in E. coli BL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability.

Show MeSH

Related in: MedlinePlus

Alignment of amino acid sequences of PsGrx with the sequences of other Grxs. The displayed sequences are Pseudoalteromonas haloplanktis Grx (YP_338909), Pseudoalteromonas agarivorans Grx (WP_004588615), Pseudoalteromonas haloplanktis Grx (WP_016708488), Pseudoalteromonas undina Grx (WP_010391834), and Rheinheimera nanhaiensis Grx (WP_008217797). The shaded boxes in same color indicate identical residues. Symbols: closed triangles, cysteines (C) in the active site; pentagrams, residues involved in glutathione-binding site.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109671&req=5

fig1: Alignment of amino acid sequences of PsGrx with the sequences of other Grxs. The displayed sequences are Pseudoalteromonas haloplanktis Grx (YP_338909), Pseudoalteromonas agarivorans Grx (WP_004588615), Pseudoalteromonas haloplanktis Grx (WP_016708488), Pseudoalteromonas undina Grx (WP_010391834), and Rheinheimera nanhaiensis Grx (WP_008217797). The shaded boxes in same color indicate identical residues. Symbols: closed triangles, cysteines (C) in the active site; pentagrams, residues involved in glutathione-binding site.

Mentions: A full-length Grx gene (Genbank accession no: KF361316) from P. sp. AN178, designated PsGrx, was composed of 270 nucleotides encoding 89 amino acid residues. The calculated molecular weight and isoelectric point of PsGrx were 9.8 kDa and pH 5.2, respectively. In the amino acid sequence of PsGrx, the total number of negatively charged residues (Asp + Glu) was 10, while the total number of positively charged residues (Arg + Lys) was 8. Multiple sequence alignment revealed the conserved GSH binding site (G-site) (Lys9, Cys12, Pro13, Phe14, Gly50, Thr53, and Val54), and catalytic residues (Cys12 and Cys15) were identified in the sequence (Figure 1). The blast search in the NCBI GenBank using the deduced amino acid sequence of PsGrx revealed that it had high sequence similarity to Trx-superfamily. The amino acid sequence of PsGrx shared 85.6%, 74.4%, 74.4%, 72.2%, and 50.0% identity with P. haloplanktis TAC 125 Grx (YP_338909), P. agarivorans Grx (WP_004588615), P. haloplanktis Grx (WP_016708488), P. undina Grx (WP_010391834), and R. nanhaiensis Grx (WP_008217797), respectively. This indicated that PsGrx could be a novel Grx belonging to Trx-like superfamily.


Cloning, expression, purification, and characterization of glutaredoxin from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178.

Wang Q, Hou Y, Shi Y, Han X, Chen Q, Hu Z, Liu Y, Li Y - Biomed Res Int (2014)

Alignment of amino acid sequences of PsGrx with the sequences of other Grxs. The displayed sequences are Pseudoalteromonas haloplanktis Grx (YP_338909), Pseudoalteromonas agarivorans Grx (WP_004588615), Pseudoalteromonas haloplanktis Grx (WP_016708488), Pseudoalteromonas undina Grx (WP_010391834), and Rheinheimera nanhaiensis Grx (WP_008217797). The shaded boxes in same color indicate identical residues. Symbols: closed triangles, cysteines (C) in the active site; pentagrams, residues involved in glutathione-binding site.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109671&req=5

fig1: Alignment of amino acid sequences of PsGrx with the sequences of other Grxs. The displayed sequences are Pseudoalteromonas haloplanktis Grx (YP_338909), Pseudoalteromonas agarivorans Grx (WP_004588615), Pseudoalteromonas haloplanktis Grx (WP_016708488), Pseudoalteromonas undina Grx (WP_010391834), and Rheinheimera nanhaiensis Grx (WP_008217797). The shaded boxes in same color indicate identical residues. Symbols: closed triangles, cysteines (C) in the active site; pentagrams, residues involved in glutathione-binding site.
Mentions: A full-length Grx gene (Genbank accession no: KF361316) from P. sp. AN178, designated PsGrx, was composed of 270 nucleotides encoding 89 amino acid residues. The calculated molecular weight and isoelectric point of PsGrx were 9.8 kDa and pH 5.2, respectively. In the amino acid sequence of PsGrx, the total number of negatively charged residues (Asp + Glu) was 10, while the total number of positively charged residues (Arg + Lys) was 8. Multiple sequence alignment revealed the conserved GSH binding site (G-site) (Lys9, Cys12, Pro13, Phe14, Gly50, Thr53, and Val54), and catalytic residues (Cys12 and Cys15) were identified in the sequence (Figure 1). The blast search in the NCBI GenBank using the deduced amino acid sequence of PsGrx revealed that it had high sequence similarity to Trx-superfamily. The amino acid sequence of PsGrx shared 85.6%, 74.4%, 74.4%, 72.2%, and 50.0% identity with P. haloplanktis TAC 125 Grx (YP_338909), P. agarivorans Grx (WP_004588615), P. haloplanktis Grx (WP_016708488), P. undina Grx (WP_010391834), and R. nanhaiensis Grx (WP_008217797), respectively. This indicated that PsGrx could be a novel Grx belonging to Trx-like superfamily.

Bottom Line: Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide.Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC.It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl.

View Article: PubMed Central - PubMed

Affiliation: School of Marine and Technology, Harbin Institute of Technology, Weihai 264209, China.

ABSTRACT
Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx) with 270 nucleotides was isolated from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. Recombinant PsGrx (rPsGrx) stably expressed in E. coli BL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability.

Show MeSH
Related in: MedlinePlus