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Literature summary extracted from

  • Wang, J.; Cheng, P.; Wu, Y.; Wang, A.; Liu, F.; Pei, X.
    Discovery of a new NADPH-dependent aldo-keto reductase from Candida orthopsilosis catalyzing the stereospecific synthesis of (R)-pantolactone by genome mining (2019), J. Biotechnol., 291, 26-34 .
    View publication on PubMed

Application

EC Number Application Comment Organism
1.1.1.358 synthesis the enzyme has a potential application in the asymmetric synthesis of optically active (R)-pantothenate, synthetic method of (R)-pantothenate production through the stereoselective reduction of oxopantoyl lactone (KPL) by aldo-keto reductase (AKR). (R)-Pantolactone (PL) is a key chiral building block for the synthesis of calcium (R)-pantothenate (vitamin B5), (R)-panthenol, and (R)-pantetheine, which are used as food and feed additives, as well as ingredients in pharmaceutical and cosmetic compositions Candida orthopsilosis

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.168 expressed in Escherichia coli BL21(DE3) cells Candida orthopsilosis
1.1.1.214 expression in Escherichia coli Candida orthopsilosis
1.1.1.358 recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) Candida orthopsilosis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.214 homology modeling of structure and docking analysis. Structure reveals a triosephosphate isomerase barrel fold comprised of eight beta-strands and eight alpha-helices Candida orthopsilosis

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.1.168 Ag+ complete inhibition at 1 mM Candida orthopsilosis
1.1.1.168 Co2+ 81.9% residual activity at 1 mM Candida orthopsilosis
1.1.1.168 Cu2+ 67.8% residual activity at 1 mM Candida orthopsilosis
1.1.1.168 EDTA
-
 Candida orthopsilosis
1.1.1.168 Fe2+ 81% residual activity at 1 mM Candida orthopsilosis
1.1.1.168 Mg2+ 80.2% residual activity at 1 mM Candida orthopsilosis
1.1.1.168 Mn2+ 67.6% residual activity at 1 mM Candida orthopsilosis
1.1.1.168 Pb2+ complete inhibition at 1 mM Candida orthopsilosis
1.1.1.168 Zn2+ 71.7% residual activity at 1 mM Candida orthopsilosis
1.1.1.214 Ag+ 1 mM, complete inhibition Candida orthopsilosis
1.1.1.214 Co2+ 1 mM, 81.9% residual activity Candida orthopsilosis
1.1.1.214 Cu2+ 1 mM, 67.8% residual activity Candida orthopsilosis
1.1.1.214 EDTA 1 mM, 72.9% residual activity Candida orthopsilosis
1.1.1.214 Fe2+ 1 mM, 81% residual activity Candida orthopsilosis
1.1.1.214 Mg2+ 1 mM, 67.6% residual activity; 1 mM, 80.2% residual activity Candida orthopsilosis
1.1.1.214 additional information not inhibitory: 1 mM Ni2+ or Ca2+ Candida orthopsilosis
1.1.1.214 Pb2+ 1 mM, complete inhibition Candida orthopsilosis
1.1.1.214 Zn2+ 1 mM, 71.7% residual activity Candida orthopsilosis
1.1.1.358 Ag+ complete inhibition at 1 mM Candida orthopsilosis
1.1.1.358 Co2+
-
 Candida orthopsilosis
1.1.1.358 Cu2+
-
 Candida orthopsilosis
1.1.1.358 EDTA
-
 Candida orthopsilosis
1.1.1.358 Fe2+
-
 Candida orthopsilosis
1.1.1.358 Mg2+
-
 Candida orthopsilosis
1.1.1.358 Mn2+
-
 Candida orthopsilosis
1.1.1.358 Pb2+ complete inhibition at 1 mM Candida orthopsilosis
1.1.1.358 Zn2+
-
 Candida orthopsilosis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.168 1.3
-
 2-dehydropantolactone at pH 7.0 and 30°C Candida orthopsilosis
1.1.1.214 1.3
-
 2-dehydropantolactone pH 7, 30°C Candida orthopsilosis
1.1.1.358 additional information
-
 additional information Michaelis-Menten kintics Candida orthopsilosis
1.1.1.358 1.3
-
 2-dehydropantolactone pH 7.0, 30°C, recombinant enzyme Candida orthopsilosis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.1.1.168 additional information 1 mM Ni2+ and Ca2+ have almost no influence over the enzyme activity Candida orthopsilosis
1.1.1.358 additional information Ni2+ and Ca2+ have almost no influence on the activity of CorCPR Candida orthopsilosis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.168 2-dehydropantolactone + NADPH + H+ Candida orthopsilosis
-
 (R)-pantolactone + NADP+
-
 ?
1.1.1.168 2-dehydropantolactone + NADPH + H+ Candida orthopsilosis 90-125
-
 (R)-pantolactone + NADP+
-
 ?
1.1.1.358 2-dehydropantolactone + NADPH + H+ Candida orthopsilosis
-
 (R)-pantolactone + NADP+
-
 ?
1.1.1.358 2-dehydropantolactone + NADPH + H+ Candida orthopsilosis Co 90-125
-
 (R)-pantolactone + NADP+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.168 Candida orthopsilosis H8XA83
-
-
1.1.1.168 Candida orthopsilosis 90-125 H8XA83
-
-
1.1.1.214 Candida orthopsilosis H8XA83
-
-
1.1.1.214 Candida orthopsilosis 90-125 H8XA83
-
-
1.1.1.358 Candida orthopsilosis
-
-
-
1.1.1.358 Candida orthopsilosis Co 90-125
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.168 Ni-NTA column chromatography Candida orthopsilosis
1.1.1.358 recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis Candida orthopsilosis

Reaction

EC Number Reaction Comment Organism Reaction ID
1.1.1.358 (R)-pantolactone + NADP+ = 2-dehydropantolactone + NADPH + H+ enzyme CorCPR has a catalytic tetrad D-Y-K-H, and the detailed catalytic mechanism is clarified by molecular docking, overview Candida orthopsilosis
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.168 2-dehydropantolactone + NADPH + H+
-
 Candida orthopsilosis (R)-pantolactone + NADP+
-
 ?
1.1.1.168 2-dehydropantolactone + NADPH + H+ 100% activity, 99% conversion and more than 99% enantiomeric excess within 5 h Candida orthopsilosis (R)-pantolactone + NADP+
-
 ?
1.1.1.168 2-dehydropantolactone + NADPH + H+
-
 Candida orthopsilosis 90-125 (R)-pantolactone + NADP+
-
 ?
1.1.1.168 2-dehydropantolactone + NADPH + H+ 100% activity, 99% conversion and more than 99% enantiomeric excess within 5 h Candida orthopsilosis 90-125 (R)-pantolactone + NADP+
-
 ?
1.1.1.168 ethyl 2-oxopropanoate + NADPH + H+ 0.496% activity compared to 2-dehydropantolactone Candida orthopsilosis ethyl (2R)-2-hydroxypropanoate + NADP+
-
 ?
1.1.1.168 ethyl 2-oxopropanoate + NADPH + H+ 0.496% activity compared to 2-dehydropantolactone Candida orthopsilosis 90-125 ethyl (2R)-2-hydroxypropanoate + NADP+
-
 ?
1.1.1.168 ethyl oxo(phenyl)acetate + NADPH + H+ 3.45% activity compared to 2-dehydropantolactone Candida orthopsilosis ethyl (2R)-hydroxy(phenyl)acetate + NADP+
-
 ?
1.1.1.168 ethyl oxo(phenyl)acetate + NADPH + H+ 3.45% activity compared to 2-dehydropantolactone Candida orthopsilosis 90-125 ethyl (2R)-hydroxy(phenyl)acetate + NADP+
-
 ?
1.1.1.168 methyl 2-oxopropanoate + NADPH + H+ 0.501% activity compared to 2-dehydropantolactone Candida orthopsilosis methyl (2R)-2-hydroxypropanoate + NADP+
-
 ?
1.1.1.168 methyl oxo(phenyl)acetate + NADPH + H+ 3.94% activity compared to 2-dehydropantolactone Candida orthopsilosis methyl (2R)-hydroxy(phenyl)acetate + NADP+
-
 ?
1.1.1.168 additional information the enzyme does not reduce 4-nitrobenzaldehyde Candida orthopsilosis ?
-
-
1.1.1.168 additional information the enzyme does not reduce 4-nitrobenzaldehyde Candida orthopsilosis 90-125 ?
-
-
1.1.1.168 pyridine 3-aldehyde + NADPH + H+ 0.538% activity compared to 2-dehydropantolactone Candida orthopsilosis (pyridin-3-yl)methanol + NADP+
-
 ?
1.1.1.214 2-dehydropantolactone + NADPH + H+
-
 Candida orthopsilosis (R)-pantolactone + NADP+
-
 ?
1.1.1.358 2-dehydropantolactone + NADPH + H+
-
 Candida orthopsilosis (R)-pantolactone + NADP+
-
 ?
1.1.1.358 2-dehydropantolactone + NADPH + H+ recombinant CorCPR exhibited a high catalytic activity for the synthesis of (R)-pantolactone ((R)-PL) from 2-dehydropantolactone (KPL). The activity assay is performed using purified CorCPR and crude recombinant GDH in a single batch reaction. GDH is used to complete the enzyme-coupled NADPH regeneration system. CPR-01 form Candida orthopsilosis Co 90-125 exhibits excellent enantioselectivity (enantiomeric excess of over 99%). CorCPR exhibits the highest activity and stereoselectivity toward KPL Candida orthopsilosis (R)-pantolactone + NADP+
-
 ?
1.1.1.358 2-dehydropantolactone + NADPH + H+
-
 Candida orthopsilosis Co 90-125 (R)-pantolactone + NADP+
-
 ?
1.1.1.358 2-dehydropantolactone + NADPH + H+ recombinant CorCPR exhibited a high catalytic activity for the synthesis of (R)-pantolactone ((R)-PL) from 2-dehydropantolactone (KPL). The activity assay is performed using purified CorCPR and crude recombinant GDH in a single batch reaction. GDH is used to complete the enzyme-coupled NADPH regeneration system. CPR-01 form Candida orthopsilosis Co 90-125 exhibits excellent enantioselectivity (enantiomeric excess of over 99%). CorCPR exhibits the highest activity and stereoselectivity toward KPL Candida orthopsilosis Co 90-125 (R)-pantolactone + NADP+
-
 ?
1.1.1.358 additional information the aldo-keto reductase (AKR) can catalyze ketopantoyl lactone to optically active (R)-pantolactone. A putative catalytic mechanism is proposed that Tyr63 acts as a general acid. No activity of recombinant CorCPR with typical AKR substrates such as p-nitrobenzaldehyde. Other alpha-oxoesters are not the optimal substrates of CorCPR due to the relatively low activities and low stereoselectivities Candida orthopsilosis ?
-
-
1.1.1.358 additional information the aldo-keto reductase (AKR) can catalyze ketopantoyl lactone to optically active (R)-pantolactone. A putative catalytic mechanism is proposed that Tyr63 acts as a general acid. No activity of recombinant CorCPR with typical AKR substrates such as p-nitrobenzaldehyde. Other alpha-oxoesters are not the optimal substrates of CorCPR due to the relatively low activities and low stereoselectivities Candida orthopsilosis Co 90-125 ?
-
-

Subunits

EC Number Subunits Comment Organism
1.1.1.168 ? x * 37000, His6-tagged enzyme, SDS-PAGE Candida orthopsilosis
1.1.1.214 ? x * 37000, SDS-PAGE and calculated from sequence Candida orthopsilosis
1.1.1.358 ? x * 37000, about, recombinant His6-tagged enzyme, sequence calculation and SDS-PAGE Candida orthopsilosis

Synonyms

EC Number Synonyms Comment Organism
1.1.1.168 AKR
-
 Candida orthopsilosis
1.1.1.168 NADPH-dependent aldo-keto reductase
-
 Candida orthopsilosis
1.1.1.214 CORT_0G03830
-
 Candida orthopsilosis
1.1.1.358 conjugated polyketone reductase
-
 Candida orthopsilosis
1.1.1.358 CorCPR
-
 Candida orthopsilosis
1.1.1.358 CPR-01
-
 Candida orthopsilosis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.168 40
-
-
 Candida orthopsilosis
1.1.1.214 40
-
-
 Candida orthopsilosis
1.1.1.358 40
-
 recombinant enzyme Candida orthopsilosis

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.1.1.168 30 55 the activity remains above 50% of the maximum activity between 30 and 55°C Candida orthopsilosis
1.1.1.358 30 55 over 50% of maximal activity within this range Candida orthopsilosis

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.1.1.168 40 50 the recombinant enzyme exhibits high thermal stability with half-lives of 6.3 h, 3.7 h and 52 min at 40, 45 and 50°C, respectively Candida orthopsilosis
1.1.1.358 40 50 purified recombinant His6-tagged enzyme, pH 7.0, half-lives of the enzyme at 40, 45 and 50°C are 6.3 h, 3.7 h and 52 min, respectively Candida orthopsilosis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.168 6 7
-
 Candida orthopsilosis
1.1.1.214 6.5
-
-
 Candida orthopsilosis
1.1.1.358 6.5
-
 recombinant enzyme Candida orthopsilosis

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.1.1.168 6 8 more than 40% activity between pH 6.0 and 8.0 Candida orthopsilosis
1.1.1.214 6 8 more than 40% of maximum activity Candida orthopsilosis
1.1.1.358 6 8 over 40% of maximal activity within this range Candida orthopsilosis

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.168 NADPH
-
 Candida orthopsilosis
1.1.1.358 NADPH NADPH is embedded in the coenzyme binding groove in an extended conformation, and the nicotinamide ring positioned at the bottom of the substrate binding cavity. The adenosine 2'-phosphate group is bound to the side chains of Lys28, Ser263 and Arg267 of CorCPR Candida orthopsilosis

General Information

EC Number General Information Comment Organism
1.1.1.358 evolution enzyme CorCPR belongs to the AKR3C family of AKR superfamily, it also contains the conserved GXGT/SX motif of the AKR superfamily Candida orthopsilosis
1.1.1.358 additional information docking analysis and structure homology modeling of CorCPR, the crystal structure of CPR-C2 from Candida parapsilosis strain IFO 0708 (PDB ID 3VXG, resolution 1.7 A) is used as the model structure, overview. KPL is docked into the substrate binding pocket. The model predicts that the C3 carbonyl oxygen of KPL forms hydrogen bond with the side chains of Tyr63, and the alpha-amino group of Thr27 also forms a hydrogen bond with C2 carbonyl oxygen. The catalytic tetrads of CorCPR are Asp58, Tyr63, Lys88 and His125. Tyr63 acts as a general acid, and His125 facilitates proton donation, the phenolic hydroxy group of Tyr63 provide general acid catalytic assistance to carbonyl group of KPL, and residues Asp 58 and Tys88 are responsible for the hydrogen transfer. The stereospecificity of CorCPR is respected to the pro-R hydrogen at C4 of the nicotinamide ring and the pseudo re-side attack of the hydride on the carbonyl group. In addition, the formation of hydrogen bond between Thr27 and the C2 carbonyl oxygen of KPL plays an important role in substrate recognition Candida orthopsilosis