Abstract
We show that the open reading frame in theThermotoga maritimagenome tentatively identified as thepcmgene (R. V. Swansonet al., J. Bacteriol.178, 484–489, 1996) does indeed encode a proteinl-isoaspartate (d-aspartate)O-methyltransferase (EC 2.1.1.77) and that this protein repair enzyme displays several novel features. We expressed the 317 amino acid pcm gene product of this thermophilic bacterium inEscherichia colias a fusion protein with an N-terminal 20 residue hexa-histidine-containing sequence. This protein contains a C-terminal domain of approximately 100 residues not previously seen in this enzyme from various prokaryotic or eukaryotic species and which does not have sequence similarity to any other entry in the GenBank databases. The C-terminal region appears to be required for enzymatic function as no activity is detected in two recombinant constructs lacking this domain. Sedimentation equilibrium analysis indicated that the enzyme is monomeric in solution. TheKmvalues for measured for peptide and protein substrates were found to be intermediate between those of the high-affinity human enzyme and those of the lower-affinity wheat, nematode, andE. colienzymes. The enzyme was extremely heat stable, with no loss of activity after 60 min at 100°C. Enzyme activity was observed at temperatures as high as 93°C with an optimal activity of 164 nmol/min/mg protein at 85°C. This activity is approximately 18-fold higher than the maximal activities of mesophilic homologs at 37°C. These data suggest that theThermotogaenzyme has unique features for initiating repair in damaged proteins containingl-isoaspartyl residues at elevated temperatures.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.