1.14.11.56: L-proline cis-4-hydroxylase

This is an abbreviated version!
For detailed information about L-proline cis-4-hydroxylase, go to the full flat file.

Reaction

L-proline
+
2-oxoglutarate
+
O2
=
cis-4-hydroxy-L-proline
+
succinate
+
CO2

Synonyms

cis-P4H, MlP4H, mlr6283, R03107, SmP4H

ECTree

     1 Oxidoreductases
         1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen
             1.14.11 With 2-oxoglutarate as one donor, and incorporation of one atom of oxygen into each donor
                1.14.11.56 L-proline cis-4-hydroxylase

Engineering

Engineering on EC 1.14.11.56 - L-proline cis-4-hydroxylase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
V95A
no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
V95A/V97A
no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
V95W
mutation significantly reduces the amount of cis-3-hydroxy-L-pipecolic acid (<5%) production compared with that of cis-5-hydroxy-L-pipecolic acid
V97A
2.5fold increase in production of cis-5-hydroxy-L-pipecolic acid, ratio of cis-5/cis-3 products is similar to wild-type
V97C
4fold increase in production of cis-5-hydroxy-L-pipecolic acid, ration of §-cis/5cis products is similar to wild-type
V97F
the cis-5/cis-3 product ratio improves from 1.4 for the wild-type enzyme to 5.3, the total amount of product is similar to wild-type
V97F/V95W
significantly improved regioselectivity toward cis-5-hydroxy-L-pipecolic acid
V97F/V95W/E114G
significantly improved regioselectivity toward cis-5-hydroxy-L-pipecolic acid and high productivity
V97Y
the cis-5/cis-3 product ratio improves from 1.4 for the wild-type enzyme to 9.0, the total amount of product decreases
V95A
-
no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
-
V95A/V97A
-
no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
-
V97A
-
2.5fold increase in production of cis-5-hydroxy-L-pipecolic acid, ratio of cis-5/cis-3 products is similar to wild-type
-
V97C
-
4fold increase in production of cis-5-hydroxy-L-pipecolic acid, ration of §-cis/5cis products is similar to wild-type
-
V97F
-
the cis-5/cis-3 product ratio improves from 1.4 for the wild-type enzyme to 5.3, the total amount of product is similar to wild-type
-
V95A
-
site-directed mutagenesis, the mutant does not show any increase in hydroxylation activity nor any improvement in the cis-5/cis-3 ratio compared to the wild-type enzyme
V95A/V97A
-
site-directed mutagenesis, the mutant does not show any increase in hydroxylation activity nor any improvement in the cis-5/cis-3 ratio compared to the wild-type enzyme
V97A
-
site-directed mutagenesis, the mutant shows increased activity and production of cis-4-hydroxy-L-proline
V97C
-
site-directed mutagenesis
V97F
-
site-directed mutagenesis, the mutant shows improved regioselectivity of hydroxylation, the cis-5/cis-3 ratio improves from 1.4 for the wild-type enzyme to 5.3 for the mutant, the increase in activity is similar compared to mutant V97A, the V97F mutant demonstrates higher selectivity of C5-hydroxylation
V97F/V95W
-
site-directed mutagenesis, the mutant shows improved regioselectivity and increased activity of hydroxylation
V97F/V95W/E114G
-
site-directed mutagenesis, protein engineering of L-proline cis-4-hydroxylase based on the X-ray crystal structure leading to refined regio- and stereoselective hydroxylation of L-pipecolic acid, the engineered mutant enzyme produces 96% cis-5-hydroxypipecolate and 4% cis-3-hydroxypipecolate while the wild-type produces 60% cis-5-hydroxypipecolate and 40% cis-3-hydroxypipecolate. A structure homology model of the SmP4H triple mutant V97F/V95W/E114G is constructed based on the MlP4H crystal structure. addition of the E114G mutation improves the activity approximately 2fold compared to double mutant V97F/V95W. The triple mutant shows the highest growth and productivity of cis-5-hydroxy-L-pipecolate in a regioselective manner
V97Y
-
site-directed mutagenesis, the mutant shows improved regioselectivity of hydroxylation, the cis-5/cis-3 ratio improves from 1.4 for the wild-type enzyme to 9.0 for the mutant, the increase in activity is decreased compared to mutant V97F