Production and Characterization of Recombinant Wild Type Uricase from Indonesian Coelacanth (L. menadoensis) and Improvement of Its Thermostability by In Silico Rational Design and Disulphide Bridges Engineering

Yainoy, Sakda; Phuadraksa, Thanawat; Wichit, Sineewanlaya; Sompoppokakul, Maprang; Songtawee, Napat; Prachayasittikul, Virapong; Isarankura‐Na‐Ayudhya, Chartchalerm

doi:10.3390/ijms20061269

Cited by 15 publications

(8 citation statements)

References 49 publications

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“…4 ). In attempt to further stabilize An96, we inserted disulfide bridges into the protein ( Craig and Dombkowski 2013 ; Yainoy et al 2019 ). One variant (Coel) had amino acid replacements at M25C and N287C, while a different variant (DbD3) had replacements at A223C and A130C.…”

Section: Resultsmentioning

confidence: 99%

Phylogenetic Articulation of Uric Acid Evolution in Mammals and How It Informs a Therapeutic Uricase

Hoshino

Tran

et al. 2021

Molecular Biology and Evolution

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The role of uric acid during primate evolution has remained elusive ever since it was discovered over 100 years ago that humans have unusually high levels of the small molecule in our serum. It has been difficult to generate a neutral or adaptive explanation in part because the uricase enzyme evolved to become a pseudogene in apes thus masking typical signals of sequence evolution. Adding to the difficulty is a lack of clarity on the functional role of uric acid in apes. One popular hypothesis proposes that uric acid is a potent antioxidant that increased in concentration to compensate for the lack of vitamin C synthesis in primate species ∼65 Ma. Here, we have expanded on our previous work with resurrected ancient uricase proteins to better resolve the reshaping of uricase enzymatic activity prior to ape evolution. Our results suggest that the pivotal death-knell to uricase activity occurred between 20 and 30 Ma despite small sequential modifications to its catalytic efficiency for the tens of millions of years since primates lost their ability to synthesize vitamin C, and thus the two appear uncorrelated. We also use this opportunity to demonstrate how molecular evolution can contribute to biomedicine by presenting ancient uricases to human immune cells that assay for innate reactivity against foreign antigens. A highly stable and highly catalytic ancient uricase is shown to elicit a lower immune response in more human haplotypes than other uricases currently in therapeutic development.

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Section: Resultsmentioning

confidence: 99%

Phylogenetic Articulation of Uric Acid Evolution in Mammals and How It Informs a Therapeutic Uricase

Hoshino

Tran

et al. 2021

Molecular Biology and Evolution

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“…3A, supporting the hypothesis that H 2 O is the second nucleophile. We verified this finding using an H 2 18 O isotope-labeling assay, which directly verified H 2 O as the second nucleophile. The data showed that the m/z ratio of all characteristic 2-methylbutyric acid fragment ions containing hydroxyl groups increased by 2, compared with a control assay with normal H 2 16 O (Fig.…”

Section: Mechanism and Engineering Of Lovastatin Esterase Pcestmentioning

confidence: 55%

“…Our previous work showed that the main problems with PcEST are its poor soluble expression and thermostability (9). The highly flexible regions on protein surfaces sometimes affect the solubility of the proteins and/or reduce their thermostability (17,18), because they might enhance the entropy during protein unfolding by increasing the numbers of unfolded conformations (19). In the PcEST structure, the long loop in R2 and the antiparallel ␤-sheet that may participate in the conformation stabilization of R2 helix showed high flexibility with higher structural B factors.…”

Section: Structure-guided Engineering Of Pcestmentioning

confidence: 99%

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Structural insights into the catalytic mechanism of lovastatin hydrolase

Liang

Lü

2019

J. Biol. Chem.

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The lovastatin hydrolase PcEST from the fungus Penicillium chrysogenum exhibits enormous potential for industrial-scale applications in single-step production of monacolin J, the key precursor for synthesis of the cholesterol-lowering drug simvastatin. This enzyme specifically and efficiently catalyzes the conversion of lovastatin to monacolin J but cannot hydrolyze simvastatin. Understanding the catalytic mechanism and the structure–function relationship of PcEST is therefore important for further lovastatin hydrolase screening, engineering, and commercial applications. Here, we solved four X-ray crystal structures, including apo PcEST (2.3 Å), PcEST in complex with monacolin J (2.48 Å), PcEST complexed with the substrate analog simvastatin (2.4 Å), and an inactivated PcEST variant (S57A) with the lovastatin substrate (2.3 Å). Structure-based biochemical analyses and mutagenesis assays revealed that the Ser57 (nucleophile)–Tyr170 (general base)–Lys60 (general acid) catalytic triad, the hydrogen-bond network (Trp344 and Tyr127) around the active site, and the specific substrate-binding tunnel together determine efficient and specific lovastatin hydrolysis by PcEST. Moreover, steric effects on nucleophilic attack caused by the 2′,2-dimethybutyryl group of simvastatin resulted in no activity of PcEST on simvastatin. On the basis of structural comparisons, we propose several indicators to define lovastatin esterases. Furthermore, using structure-guided enzyme engineering, we developed a PcEST variant, D106A, having improved solubility and thermostability, suggesting a promising application of this variant in industrial processes. To our knowledge, this is the first report describing the mechanism and structure–function relationship of lovastatin hydrolase and providing insights that may guide rapid screening and engineering of additional lovastatin esterase variants.

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“…Isolation of uricase from green bean leaves [ [4]], Pseudomonas aeruginosa [ [5]], Streptomyces exfoliates UR10 from agricultural waste [ [6]], Bacillus subtilis strain SP6 [ [7]], Bacillus cereus SKIII [ [8]], Aspergillus welwitschiae strain 1-4 [ [9]]. Isolation of the uricase from the liver of rainbow fish, mackerel, trout, catfish, shark and tilapia [ [10]], Indonesian coelacanth [ [11]], camel liver [ [12]], and bovine kidney [ [13]]. Based on previous studies, this study will isolate and characterize the uricase from chicken (Gallus gallus domesticus) liver which are broiler and native chicken liver.…”

Section: Introductionmentioning

confidence: 99%

Isolation and Characterization of Uricase Produced from Chicken Liver

Handayani¹,

Naja²,

Joenata³

et al. 2022

jbiome

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Uricase is an enzyme that degrades uric acid into allantoin. One of the uricase sources is obtained from chicken species (Gallus gallus domesticus) liver which are broiler and native chicken. This study aims to determine the maximum uricase activity in broiler and native chicken liver. The uricase activity was obtained by measuring the uric acid concentration as uricase substrate using spectrophotometric method and wavelength at 291 nm. Uricase isolation was carried out into extraction process, ammonium sulfate fractionation (0-60% saturation of ammonium sulfate), and dialysis. During isolation process, centrifugation speed was also optimized to obtain the maximum uricase crude extract and uricase activity. The molecular weight of uricase was also determined by SDS PAGE. The result showed that the highest uricase activity remained using centrifugation speed of 15,000 rpm. The optimum uricase fraction for broiler chicken liver was obtained at 20-40% saturation of ammonium sulfate with uricase activity was 1.854 x 10-2 U/mg, and the uricase fraction for native chicken liver was obtained at 40-60% saturation of ammonium sulfate with uricase activity was 2.496 x 10-2 U/mg. The optimum fraction for uricase production and isolation is carried out to the dialysis process. The optimum uricase activity of broiler chicken liver crude extract was 4.921 x 10-4 U/mg, the uricase fraction was 3.989 x 10-3 U/mg, and the dialysate was 5.120 x 10-3 U/mg. While the native chicken liver crude extract was 2.980 x 10-4 U/mg, the uricase fraction was1.415 x 10-2 U/mg, and the dialysate was 1.753 x 10-2 U/mg. The molecular weight of the uricase was around 35 kDA according to the SDS PAGE result.

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Production and Characterization of Recombinant Wild Type Uricase from Indonesian Coelacanth (L. menadoensis) and Improvement of Its Thermostability by In Silico Rational Design and Disulphide Bridges Engineering

Cited by 15 publications

References 49 publications

Phylogenetic Articulation of Uric Acid Evolution in Mammals and How It Informs a Therapeutic Uricase

Phylogenetic Articulation of Uric Acid Evolution in Mammals and How It Informs a Therapeutic Uricase

Structural insights into the catalytic mechanism of lovastatin hydrolase

Isolation and Characterization of Uricase Produced from Chicken Liver

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