Molecular basis for thermal properties of Streptomyces thermovulgaris fumarase C hinge at hydrophilic amino acids R163, E170 and S347.
Industrially, the use of high temperatures (40-60 degrees C) in the L: -malate production process could result in rapid inactivation of the mesophilic fumarases, warranting constant replenishment of the biocatalyst. Thus, a thermostable fumarase C that is active and stable at high temperatures would be ideal. Biochemical studies using recombinant fumarase C from thermophilic Streptomyces thermovulgaris (stFUMC) indicated that it was optimally active at 50 degrees C and highly stable even after 24 h of incubation at 40 degrees C. The same gene from mesophilic Streptomyces coelicolor (scfumC) was also cloned and expressed as soluble proteins for comparison in thermal properties of both enzymes. In contrast to stFUMC, scFUMC exhibited a lower temperature optima of 30 degrees C and was rapidly denatured at 50 degrees C. The specific activity of stFUMC was also higher than that of scFUMC by 20-fold. After primary sequence comparison, three hydrophilic amino acid residues, R163, E170 and S347, were forged into the thermolabile scFUMC either singly or in combination for the investigation of their contributions in the thermal properties of the mutant enzymes. Of the mutants studied, the A347S scFUMC mutant resulted in the highest increase in optimum temperature of 10 degrees C and a fourfold enhancement in specific activity. G163R/G170E and G163R/G170E/A347S scFUMC mutants are more thermostable than wild-type scFUMC. These findings support stFUMC as a highly efficient, thermostable fumarase C with industrial potential and suggest that R163, E170 and S347 are involved in the enhancement of thermal properties in fumarase C.
Lin
Wenjie
W
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore, 117597, Singapore.
Chan
Maurice
M
Goh
Liuh-Ling
LL
Sim
Tiow-Suan
TS
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005649:Fumarate Hydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006859:Hydrogen / Q000737:chemistry*; D007218:Industrial Microbiology / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D048372:Streptomyces coelicolor / Q000201:enzymology / Q000235:genetics
D000596:Amino Acids; D006859:Hydrogen; C095842:fumarase C; D005649:Fumarate Hydratase