Biochemical characterization of an esterase from Clostridium acetobutylicum with novel GYSMG pentapeptide motif at the catalytic domain

By Vijayalakshmi Nagaroor, Sathyanarayana N. Gummadi

March 24, 2020

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Title

Biochemical characterization of an esterase from Clostridium acetobutylicum with novel GYSMG pentapeptide motif at the catalytic domain

Author

Vijayalakshmi Nagaroor, Sathyanarayana N. Gummadi

Year

2019

Journal

Journal of Industrial Microbiology & Biotechnology

Abstract

Gene CA_C0816 codes for a serine hydrolase protein from Clostridium acetobutylicum (ATCC 824) a member of hormone-sensitive lipase of lipolytic family IV. This gene was overexpressed in E. coli strain BL21and purified using Ni2+–NTA affinity chromatography. Size exclusion chromatography revealed that the protein is a dimer in solution. Optimum pH and temperature for recombinant Clostridium acetobutylicum esterase (Ca-Est) were found to be 7.0 and 60 °C, respectively. This enzyme exhibited high preference for p-nitrophenyl butyrate. KM and kcat/KM of the enzyme were 24.90 µM and 25.13 s−1 µM−1, respectively. Sequence analysis of Ca-Est predicts the presence of catalytic amino acids Ser 89, His 224, and Glu 196, presence of novel GYSMG conserved sequence (instead of GDSAG and GTSAG motif), and undescribed variation of HGSG motif. Site-directed mutagenesis confirmed that Ser 89 and His 224 play a major role in catalysis. This study reports that Ca-Est is hormone-sensitive lipase with novel GYSMG pentapeptide motif at a catalytic domain.

Full Article

https://link.springer.com/article/10.1007/s10295-019-02253-8

Instrument

J-815

Keywords

Circular dichroism, Thermal stability, Secondary structure, Protein folding, Biochemistry