Improvement of 2'-hydroxybiphenyl-2-sulfinate desulfinase, an enzyme involved in the dibenzothiophene desulfurization pathway, from Rhodococcus erythropolis KA2-5-1 by site-directed mutagenesis.
In the microbial dibenzothiophene desulfurization pathway, 2'-hydroxybiphenyl-2-sulfinate is converted to 2-hydroxybiphenyl and sulfinate by desulfinase (DszB) at the last step, and this reaction is rate-limiting for the whole pathway. The catalytic activity and thermostability of DszB were enhanced by the two amino acid substitutions. Based on information on the 3-D structure of DszB and a comparison of amino acid sequences between DszB and reported thermophilic and thermostable homologs (TdsB and BdsB), two amino acid residues, Tyr63 and Gln65, were selected as targets to mutate and improve DszB. These two residues were replaced by several amino acids, and the promising mutant enzymes were purified and their properties were examined. Among the wild-type and mutant enzymes, Y63F had higher catalytic activity but similar thermostability, and Q65H showed higher thermostability but less catalytic activity and affinity for the substrate. To compensate for these drawbacks, the double mutant enzyme Y63F-Q65H was purified and its properties were investigated. This mutant enzyme showed higher thermostability without loss of catalytic activity or affinity for the substrate. These superior properties of the mutant enzyme have also been confirmed with resting cells harboring the mutant gene.
Ohshiro
Takashi
T
Department of Biotechnology, Tottori University, Japan.
Ohkita
Ryo
R
Takikawa
Takeshi
T
Manabe
Masanori
M
Lee
Woo Cheol
WC
Tanokura
Masaru
M
Izumi
Yoshikazu
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D012240:Rhodococcus / Q000201:enzymology*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D005973:Glutamine; D014443:Tyrosine; C113832:2-(2-hydroxybiphenyl)-benzenesulfinate desulfinase; D050862:Oxidoreductases Acting on Sulfur Group Donors