Structural and catalytic alteration of sarcosine oxidase through reconstruction with coenzyme-like ligands

Xin, Yu; Zheng, Mengling; Wang, Qing; Lu, Liushen; Zhang, Ling; Tong, Yanjun; Wu, Wei

doi:10.1016/j.molcatb.2017.01.011

Cited by 9 publications

(19 citation statements)

References 26 publications

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“…Overall, the SPα‐SOX displays a relatively high energy barrier to denaturation, with a measured temperature of melting ( T m ) of 59.3 ± 0.3°C and 63.2 ± 0.02°C for the two domain, respectively (Figure 3b). The reported T m of native sarcosine oxidase is 64.0°C, 43,44 in line with our data, for this reason, we assigned the first endothermal peak of the thermogram to P450 Spα domain ( T m : 59.3°C) and the second endothermal peak to MSOX domain ( T m : 63.2°C) (Figure 3b). To our knowledge, the melting temperature of P450 Spα has not been reported yet.…”

Section: Resultssupporting

confidence: 88%

Design of a H₂O₂‐generating P450_SPα fusion protein for high yield fatty acid conversion

et al. 2022

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Sphingomonas paucimobilis' P450SPα (CYP152B1) is a good candidate as industrial biocatalyst. This enzyme is able to use hydrogen peroxide as unique cofactor to catalyze the fatty acids conversion to α‐hydroxy fatty acids, thus avoiding the use of expensive electron‐donor(s) and redox partner(s). Nevertheless, the toxicity of exogenous H2O2 toward proteins and cells often results in the failure of the reaction scale‐up when it is directly added as co‐substrate. In order to bypass this problem, we designed a H2O2 self‐producing enzyme by fusing the P450SPα to the monomeric sarcosine oxidase (MSOX), as H2O2 donor system, in a unique polypeptide chain, obtaining the P450SPα‐polyG‐MSOX fusion protein. The purified P450SPα‐polyG‐MSOX protein displayed high purity (A417/A280 = 0.6) and H2O2‐tolerance (kdecay = 0.0021 ± 0.000055 min−1; ΔA417 = 0.018 ± 0.001) as well as good thermal stability (Tm: 59.3 ± 0.3°C and 63.2 ± 0.02°C for P450SPα and MSOX domains, respectively). The data show how the catalytic interplay between the two domains can be finely regulated by using 500 mM sarcosine as sacrificial substrate to generate H2O2. Indeed, the fusion protein resulted in a high conversion yield toward fat waste biomass‐representative fatty acids, that is, lauric acid (TON = 6,800 compared to the isolated P450SPα TON = 2,307); myristic acid (TON = 6,750); and palmitic acid (TON = 1,962).

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Section: Resultssupporting

confidence: 88%

Design of a H₂O₂‐generating P450_SPα fusion protein for high yield fatty acid conversion

et al. 2022

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“…(GenBank: AY626822.2, BSOX ) was also recombinantly expressed and characterized. Despite having 36.6% sequence identity with TrSOX, BSOX showed poor stability ( T m at 54 °C, and the half-life at 60 °C was <10 min); however, it could be expressed in Escherichia coli cells at a relatively high level in a shaking flask (approximately 8–10 mg/g wet pellet), which could be further optimized for industrial scale production …”

Section: Introductionmentioning

confidence: 99%

Aggregation Interface and Rigid Spots Sustain the Stable Framework of a Thermophilic N-Demethylase

Sun

Zhu

et al. 2023

J. Agric. Food Chem.

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Enzymes from thermophilic microorganisms usually show high thermostability, which is of great potential in industrial application; to understand the structural logic of these enzymes is helpful for the construction of robust biocatalysts. In this study, based on the crystal structure of an N-demethylase�TrSOX�with outstanding thermostability from Thermomicrobium roseum, substitutions were introduced on the aggregation interface and rigid spots to reduce the aggregation ratio and the rigidity. Four substitutions on the aggregation interface�V162S, M308S, F170S, and V306S�considerably reduced the thermostability and slightly enhanced the catalytic efficiency. In addition, the thermostable framework was considerably disrupted in several multiple P → G substitutions in several local motifs (P129G/P134G, P237G/P259G, and P259G/P276G). These structural fluctuations were in good accordance with whole-structure or partial root-mean-square deviation, radius of gyration H-bonds, and solvent-accessible surface area values in molecular dynamics simulation. Furthermore, these key spots were introduced into an unstable homolog from Bacillus sp., resulting in a dramatical increase in the half-life at 60 °C from <10 to 1440 min. These results could help understand the natural stable framework of thermophilic enzymes, which could be references for the construction of robust enzymes in industrial applications.

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“…co-solvents), or salinity for example [ 126 ]. In other instances, genome mining of sequenced organisms or related species highlighted uncharacterized variants of well-known enzymes such as sarcosine oxidase [ 127 ], or β-agarase [ 108 ].…”

Section: Role Of Systems Biology In Addressing the Challenges Around The Formation Of Inclusion Bodiesmentioning

confidence: 99%

“…The conserved regions for a given enzyme were compared across species using alignment programs such as EMBL-EBI’s Clustal Omega [ 130 – 133 ] or other algorithms such as Needleman–Wunsch Global Align Nucleotide Sequence [ 68 , 134 ]; these conserved domains, such as active site residues, assisted researchers to interpret and predict an enzyme’s function [ 135 ]. Beyond these methods, we observed the use of SWISS-MODEL [ 127 , 135 – 137 ] and PyMOL [ 42 , 138 ] for comparative 3D modelling of the evolutionary relationship between target proteins and the prediction of substrate-enzyme docking interactions. It is expected that this methodology for predictive structural modelling of new DtE enzymes will be highly influenced by innovations incorporating novel neural network architectures such as AlphaFold [ 139 ].…”

Section: Role Of Systems Biology In Addressing the Challenges Around The Formation Of Inclusion Bodiesmentioning

confidence: 99%

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

2021

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Recombinant enzyme expression in Escherichia coli is one of the most popular methods to produce bulk concentrations of protein product. However, this method is often limited by the inadvertent formation of inclusion bodies. Our analysis systematically reviews literature from 2010 to 2021 and details the methods and strategies researchers have utilized for expression of difficult to express (DtE), industrially relevant recombinant enzymes in E. coli expression strains. Our review identifies an absence of a coherent strategy with disparate practices being used to promote solubility. We discuss the potential to approach recombinant expression systematically, with the aid of modern bioinformatics, modelling, and ‘omics’ based systems-level analysis techniques to provide a structured, holistic approach. Our analysis also identifies potential gaps in the methods used to report metadata in publications and the impact on the reproducibility and growth of the research in this field.

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Structural and catalytic alteration of sarcosine oxidase through reconstruction with coenzyme-like ligands

Cited by 9 publications

References 26 publications

Design of a H₂O₂‐generating P450_SPα fusion protein for high yield fatty acid conversion

Design of a H₂O₂‐generating P450_SPα fusion protein for high yield fatty acid conversion

Aggregation Interface and Rigid Spots Sustain the Stable Framework of a Thermophilic N-Demethylase

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

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Structural and catalytic alteration of sarcosine oxidase through reconstruction with coenzyme-like ligands

Cited by 9 publications

References 26 publications

Design of a H2O2‐generating P450SPα fusion protein for high yield fatty acid conversion

Design of a H2O2‐generating P450SPα fusion protein for high yield fatty acid conversion

Aggregation Interface and Rigid Spots Sustain the Stable Framework of a Thermophilic N-Demethylase

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

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Design of a H₂O₂‐generating P450_SPα fusion protein for high yield fatty acid conversion

Design of a H₂O₂‐generating P450_SPα fusion protein for high yield fatty acid conversion