A cold-adapted tyrosinase with an abnormally high monophenolase/diphenolase activity ratio originating from the marine archaeon Candidatus Nitrosopumilus koreensis

Abstract

The acidic and cold-adapted tyrosinase-CNK, as a new type of tyrosinase, could expand potential applications of tyrosinases including the production of catechols through minimizing unwanted self-oxidation and the modification of existing materials at low temperature.

A tyrosinase, mTyr-CNK, that is functionally available as a monophenol monooxygenase

“…50% of total protein, based on SDS-PAGE image analysis) in culture without chaperones at 20 °C and 200 rpm after the culture was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), despite the simultaneous formation of insoluble inclusion bodies (Fig. 2 ); in contrast, GroES and GroEL chaperones were required for the functional expression of tyrosinase-CNK in a previous study 18 . Finally, mTyr-CNK was simply purified by 6xHis-tag affinity chromatography to greater than 95% purity.…”

“…Most impressively, mTyr-CNK exhibited the significantly high monophenolase/diphenolase activity ratio ( V max mono/ V max di) of 3.83, and the k cat /K m value of the monophenolase reaction was only slightly lower (87%) than that of the diphenolase reaction (Table 1 ), whereas the oxidation of catechols typically proceeds two orders of magnitude faster than the hydroxylation of monophenols 17 , 28 . The activity ratio ( V max mono/ V max di) of mTyr-CNK was approximately two-fold higher than that of tyrosinase-CNK based on the previously reported kinetic parameters of tyrosinase-CNK: k cat = 4.3 ± 1.08 (s −1 ), K m = 9.2 ± 2.4 (mM), and k cat / K m = 0.47 (mM −1 ·s −1 ) for L -tyrosine and k cat = 2.2 ± 0.47 (s −1 ), K m = 2.6 ± 0.56 (mM), and k cat / K m = 0.85 (mM −1 ·s −1 ) for L -DOPA at pH 6 and 25 °C 18 . The turnover number and binding affinity of the enzyme for L -tyrosine were higher than those for L -DOPA, possibly resulting from a reorganization of the active site and the relatively high accessibility of the active site to substrates based on previously proposed structure-function correlations for other known tyrosinases 4 , 29 , although biochemical and structural investigations of mTyr-CNK are still required to account for its surprisingly high monophenolase/diphenolase activity ratio.…”

“…Interestingly, a homology model predicted by I-TASSER revealed that tyrosinase-CNK also has a C-terminal extension (Fig. 1a,b ); similar extensions have been known to prevent the activation of fungal and plant tyrosinases 18 – 20 . Although tyrosinase-CNK showed a high catalytic efficiency for L -tyrosine that was comparable to that of other known tyrosinases and phylogenetic analysis indicated that it was distinct from other known tyrosinases, we still expect that removal of the C-terminal extension in tyrosinase-CNK has great potential to significantly improve the enzyme’s catalytic property.…”

“…Moreover, recombinants could be efficiently modified in a directed manner by introducing/deleting/changing amino acid sequences at the genetic level 14 – 17 . We recently produced a psychrophilic tyrosinase, tyrosinase-CNK, from the marine archaeon Candidatus Nitrosopumilus koreensis in E. coli by heterologous expression 18 . Interestingly, a homology model predicted by I-TASSER revealed that tyrosinase-CNK also has a C-terminal extension (Fig.…”

Design and Use of de novo Cascades for the Biosynthesis of New Benzylisoquinoline Alkaloids

“…BmTYR and RmTYR showed ap reference towards 4,giving 5 in 30-75 %yield and 2 in lower yields (15-45 %). When using purified CnTYR, the apparent kinetic parameters K m and k cat towards 4 were 1.78 mm and 31.6 s À1 (k cat,app /K m,app 1.78 10 4 s À1 m À1 )a nd the corresponding values for 6 were 3.43 mm and 55.2 s À1 (k cat,app /K m,app 1.61 10 4 s À1 m À1 ), respectively;a lthough CnTYR showed greater affinity towards 4,the k cat,app was higher with 6.The sequence identity to ar elated TYR from Candidatus Nitrosopumilus koreensis (CnkTYR) [32] is 54 %, and the K m and k cat towards 4 for this enzyme were 9.2 mm and 4.3 s À1 (k cat /K m 4.7 10 4 s À1 m À1 ). Theh igher apparent monophenolase activity with the CnTYR identified here may be due to the addition of 7.…”