The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum

QC 20180320
The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a pyridoxal-5’-phosphate (PLP)dependent enzyme. The biological activity of this enzyme requires the formation of a holo homo dimer.The operational stability of Cv-ATA is, however, low due to dimer dissociation. At the enzyme dimeric interface, two phosphate group binding cups (PGBC) are located. Each cup coordinates the phosphate group of PLP by hydrogen bonds originating from both subunits. Hypothetically, molecular coordination of phosphate groups (PLP or free inorganic phosphate) into the PGBC can affect both dimer stabilization and enzyme activity. To test this assumption, the influence of phosphate (as a functional group in PLP or as free inorganic anions) on the stability and activity of Cv-ATA was explored by various biophysical techniques. The results show that Cv-ATA has a relatively low affinity towards PLP, which results in an excess of apo dimeric enzyme after enzyme purification. Incubation of the apo dimer in buffer solution supplemented with PLP restored the active holo dimer. The addition of PLP or inorganic phosphate into the enzyme storage solutions protected Cv-ATA from both chemical and long term storage unfolding. The use of phosphate buffer leads to faster inactivation of the holo enzyme, compared to the use of HEPES buffer. These results open up for new perspectives on how to improve the stability of PLP-dependent enzymes.

NATURVETENSKAP Biologi Biokemi och molekylärbiologi hsv//swe
NATURAL SCIENCES Biological Sciences Biochemistry and Molecular Biology hsv//eng
Biocatalysis
Dimeric enzymes
PLP-dependent enzymes
Pyridoxal-5’-phosphate (PLP)
Schiff base
Bioteknologi
Biotechnology

Berglund, PerKTH,Industriell bioteknologi(Swepub:kth)u1271chh (författare)
Svedendahl Humble, MariaKTH,Industriell bioteknologi(Swepub:kth)u1mmbr9s (författare)
KTHIndustriell bioteknologi (creator_code:org_t)

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