Biochemical characterization and biocatalyticapplication of a novel L- isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis

doi:10.21203/rs.3.rs-3936523/v1

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Biochemical characterization and biocatalyticapplication of a novel L- isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis

https://doi.org/10.21203/rs.3.rs-3936523/v1

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The _L-isoleucine-4-dioxygenase can convert isoleucine into (2S,3R,4S)-4-hydroxyisoleucine (4-HIL), a naturally occurring hydroxy amino acid which is a promising compound for drug and functional food development because of its management of diabetes mellitus and reducing of total cholesterol levels in the body. Here, a novel _L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis was cloned, expressed and characterized, as one of only a few reported L-isoleucine-4-dioxygenase. Compared to functionally and structurally characterized BtIDO, recombinant RaIDO showed a higher catalytic efficeinecy (k_cat/K_m, against L-isoleucine) and stability. Homologous sequences analysis showed that shares a below 30% similarity with reported BtIDO. Notably, the RaIDO revealing a temperature tolerance range with the highest catalytic activity observed at 40℃, and an optimal reaction temperature range of 30℃-45℃. Additionally, the enzyme exhibited an optimal pH of 8.0. In a word, a new _L-isoleucine-4-dioxygenase (RaIDO)has been discovered with higher catalytic efficiency and stability, making it a strong candidate for mass production within the industry.

L-isoleucine-4-dioxygenase

diabetes

biosynthesis

high catalytic activity

temperature resistant

Amino acids are the most essential components of living organisms, they form proteins(Whitford 2013) and are involved in maintaining life activities(Benkovic and Hammes-Schiffer 2003). Amino acids with hydroxyl groups play an important role in living organisms, mainly in the maintenance of enzymatic activity, the regulation of the internal environment and the regulation of cellular activity, such as: 4-Hydroxyisoleucine (4-HIL) (Narender et al. 2006)has the unique function of stimulating glucose-induced insulin secretion in a glucose-dependent manner; it has also been shown by available clinical data to be effective in reducing total cholesterol levels in humans. 4-HIL is found only in certain plants and fungi(Zafar and Gao 2016), but the mechanism of its biosynthesis has not been elucidated(Broca et al. 2000; Sauvaire et al. 1998).

4-Hydroxylysine(Wang et al. 2023) is a highly versatile drug intermediate used in the synthesis of many important drugs such as N-methyl-D-aspartate (NMDA) antagonists and HIV protease inhibitors with anticancer, antibacterial and antiviral properties and actions.

3-Hydroxyarginine (3-OH-Arg)(Morin et al. 1998) is a key intermediate in the synthesis of viomycin, an important antibiotic used in the clinical treatment of tuberculosis.(Mao et al. 2020)

Hydroxyproline is a class of amino acids found mainly in some protein structures such as plant cell walls and animal collagen. Trans-4-hydroxy-L-proline (T4LHyp) is a component of muscarinic peptide toxins that inhibit cell growth and is used clinically to detect anti-tumour activity in cells in vivo, and biocatalytic methods have been reported for this substance(Mahlert et al. 2007).

5-Hydroxyleucine (5-Hleu)(Hibi et al. 2013) is a class of hydroxylated amino acids that has been extensively studied in recent years. It can be used as a precursor in the synthesis of drugs to produce 4-methylproline and subsequently the alkaloid bromopyrrole.

4-Hydroxyisoleucine (4-HIL) has the unique function of stimulating glucose-induced insulin secretion in a glucose-dependent manner;^{(Sauvaire et al. 1998)} it has also been shown by available clinical data to be effective in reducing total cholesterol levels in humans(Broca et al. 1999). 4-HIL is found only in certain plants and fungi(Broca et al. 2000), but the mechanism of its biosynthesis has not been elucidated(Nguyen et al. 2006).

Hydroxyamino acids are important amino acid derivatives that act as precursors or intermediates in the synthesis of various metabolites in many secondary metabolic pathways(Das et al. 2020). Due to the increasing importance of hydroxamic acids as effective drugs in the treatment of many diseases and as valuable chiral building blocks in the production of many drugs, the hydroxylation of amino acids has attracted considerable attention in the biochemical community, For example, 4-hydroxyisoleucine, 5-hydroxyamino acids such as 4-hydroxyisoleucine, 4-hydroxyisoleucine (4-HIL), a hydroxylated amino acid found in the seeds of fenugreek(Jette et al. 2009; Narender et al. 2006), can be used in the treatment of diseases such as diabetes because of its efficacy in promoting insulin secretion. It is used in the treatment of diseases such as diabetes due to its effects on insulin secretion. Available clinical trial data has also shown that 4-HIL is effective in reducing total cholesterol levels in humans. At this stage of research, the following methods have been used to produce 4-HIL: (1) chemical synthesis; (2) isolation by extraction from fenugreek seeds; and (3) bioenzymatic synthesis. Due to the lengthy and complex reaction steps of traditional chemical synthesis and isolation by extraction methods, the reaction conditions are not easily controlled and the final product produced contains a high level of by-products. Therefore, the use of bioenzymatic(Huang et al. 2016) methods(Bai et al. 2017) to catalyse the production of 4-HIL has become an important route for industrial production and a hot topic for research.

In nature, alpha-ketoglutarate-dependent mononuclear non-haem iron enzymes catalyse a variety of biochemical reactions that require Fe(II) as a metal cofactor and α-KG as a co-substrate to produce the desired product(Simmons et al. 2008), carbon dioxide, and the by-product succinate(Simmons et al. 2008).This family of enzymes is widespread in prokaryotes, eukaryotes and archaea and there are over 60 such enzymes in humans alone. Among these reactions, asymmetric hydroxylation of inactive C-atoms is an important route for the synthesis of chiral compounds, such as the synthesis of sophisticated chemicals and some drugs. This hydroxylase, which is capable of asymmetric hydroxylation reactions, is able to hydroxylate amino acids of a specific substrate in the presence of its cofactor to produce the product hydroxylated amino acid(Asuncion and Bjarne 2012). During this reaction, the cofactor Fe(II) forms a catalytically active centre with amino acids around the protein structure(Islam et al. 2018), and the co-substrate α-ketoglutarate undergoes oxidative decarboxylation to form the by-product succinate(Hibi et al. 2011), accompanied by the formation of hydroxylated amino acids. The enzymes normally catalyse the formation of hydroxylated amino acids(Wu et al. 2022).Previous studies have identified a number of valuable hydroxylated amino acids, including (2S, 4S)-5-hydroxy-leucine, 4-hydroxy-valine, N-succinyl-(2S, 3R)-3-hydroxy-leucine, cis-4-hydroxy-proline and (2S, 3R, 4S)-4-hydroxy-isoleucine, among others(Wu et al. 2022).As an amino acid biocatalyst, Fe/α-KGDs are capable of catalysing site-specific C-acid hydroxylation reactions, e.g. Previous extensive studies on the Fe(II)/αKG-dependent oxygenase family(Martinez and Hausinger 2015) have identified a number of amino acid hydroxylases with important applications, The industrial synthesis of amino acids is the major typical substrate catalysed by Fe/α-KGDs(Hibi and Ogawa 2014), which are highly enantioselective and regioselective for the carbon atoms on the amino acids when hydroxylated by Fe/α-KGDs(Hibi et al. 2016).

Finally, the gene RaIDO was obtained from Sphingomonas sphaeroides, which belongs to the Fe(II) and α-ketoglutarate-dependent oxygenase family. The purified recombinant RaIDO was used to comprehensively characterize its biochemical properties. It was found that RaIDO has higher catalytic efficiency than other enzymes that have already been reported. Additionally, it has better stability at different temperatures and pH. Furthermore, it reduces the cost of producing the enzyme and is more suitable for large-scale production.

2.1 Materials and Apparatus

L-isoleucine, 4-hydroxyisoleucine, synthesis primers were purchased from Tsingke (Beijing, China); isopropyl-β-D-1-thiogalactoside (IPTG) was purchased from Beijing BoaoTuoDa Technology Co. Plasmid extraction kit was purchased from Omega. The protein marker was purchased from Beijing PolymerMobil Biotechnology Co. Ltd. Ultrafiltration tubes were purchased from Millipore.

Ultrasonic disruptor from SONICS, USA; Analytik Jena gel imaging system from Jena, Germany; Hitachi high speed freezing centrifuge from Hitachi, Japan; ÄKTA pure from GE, USA; SIL-20A high performance liquid chromatograph from Shimadzu, Japan; Bio-Rad electrophoresis system from Bio-Rad, USA; Eppendorf PCR instrument and BioPhotometer plus from Eppendorf, Germany.

2.2 Purification of RaIDO

The plasmid pQE80L CV45-pET28b-sumo was maintained in our laboratory while the cloning host E. coli DH5α and expression host E-coli BL21 (DE3) were procured from Bomade. Yeast extract and tryptone were sourced from OXOID, and NaCl along with other standard reagents were purchased from Sinopharm Chemical Reagent Co. The RaIDO protein gene sequence was synthesized at Jin Wei Zhi and used as a template for PCR primer design. The target gene was then amplified through PCR using a 25 ul system. The PCR process involved predenaturation at 98°C for 30 s, denaturation at 98°C for 10 s, denaturation at 60°C for 10 s, denaturation at 98°C for 30 s, and denaturation at 98°C for 10 s, with annealing occurring at 60°C for 15 s and extension at 72°C for 5 s for 30 cycles. The process was then concluded with a final extension step at 72°C for 10 min. The amplified target gene underwent agarose gel electrophoresis to confirm band accuracy. Following confirmation, the target gene and vector pQE80L were digested twice by restriction endonuclease Kpn I Sac Ⅰ. The resulting digested product was purified and recovered, and subsequently ligated with solution Ⅰ at 16°C for 30 minutes. Solution 1: After ligating at 16°C for 30 minutes, the resulting plasmid will be transformed into E. coli DH5α host cells. The transformants will then be selected and subjected to double digestion verification. Samples that satisfy the criteria for the correct digestion pattern will be sent for sequencing validation.

The necessary purified proteins underwent the following procedures. The recombinant plasmid pQE80L was introduced into E.coli BL21 (DE3) cells and grown in LB medium with appropriate resistance at a temperature of 37°C and 200 rpm. Upon reaching an OD600 of 0.6–0.8, the inducer isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to attain a final concentration of 0.1 mM and induce protein expression. The culture was incubated for 12–16 hours at 16°C. Next, bacteriophage cells were harvested through centrifugation (4000 g, 15 min) and then suspended in a lysis buffer (50 mM Tris-HCl pH 8.0, 400 mM NaCl, 10% (V/V) glycerol). The cells were lysed by ultrasound. The resulting lysate was centrifuged at 13,000 g for 1 hour at 4°C. Later, proteins containing His tags were adsorbed on Ni-NTA Superflow resin for an hour. Finally, the target proteins were eluted using an elution buffer (250 mM imidazole). The recombinant proteins were purified via ion exchange and gel filtration chromatography utilizing Source Q anion exchange and SuperdexTM 200 Increase 10/300 GL gel chromatography columns. The protein concentration was assessed through the BCA method, and the protein purity was determined using the SDS-PAGE method.

2.3 Assay for enzyme activity

The HPLC technique was utilised to assess RaIDO's activity. This study employed HPLC as a means of preliminarily characterising the products resulting from amino acid hydroxylation. Technical term abbreviations have been explained upon first usage. The text is free of spelling and grammatical errors. A standard reaction system consisting of 100 uL of 20 mmol/L HEPES (pH 8.0), 0.15 mmol/L Fe(II), 2 mmol/L a-KG, ascorbic acid, 5 mmol/L isoleucine (dissolved in 20 mmol/L HEPES) as a standard substrate, and 3 umol/L purified RaIDO enzyme solution was employed to conduct the catalytic reaction. This enzyme concentration was determined, based on its optimisation for the reaction process. The RaIDO enzyme solution was utilized in the optimal concentration for the reaction process. Technical abbreviations are explained upon their first use. The reaction was conducted at room temperature (approximately 25℃) and lasted for 3 hours. At the end of the reaction, boiling was performed for termination. The enzyme activity unit is the amount of enzyme necessary to hydroxylate 1 mmol of isoleucine per minute, and the specific enzyme activity is denoted as nmol mgmin (mU/mg-1).

The protein sample was subjected to boiling and high-speed centrifugation for removal of impurities. Technical abbreviations were explained at first usage. Subsequently, 50 microlitres of the reaction solution were utilized for pre-column derivatisation with the derivatisation reagent, 2,4-dinitrofluorobenzene (DNF). For this, 0.1 millilitres of DNF was dissolved in 10 millilitres of acetonitrile (chromatographic purity). Mobile phase A was prepared using a 50 millimolar sodium acetate solution containing 0.1% triethylamine (pH 6.4, adjusted with glacial acetic acid), while mobile phase B comprised of 50% acetonitrile in water. The catalytic products were analysed via high-performance liquid chromatography (HPLC) using a SIL-20A instrument. Technical abbreviations will be defined when initially used. The separation was performed at 25°C on an XBridge C18 column (2.1 × 150 mm; Waters) with a particle size of 5 µm. The column employed was also from Waters.

Derivatization Method: To initiate the derivatization method, 50 µL of the treated catalytic reaction solution was transferred into a clean Eppendorf tube, followed by the addition of the derivatization reagent DNF (0.25 mL) and sodium bicarbonate buffer (0.25 mL). The reaction temperature was set at 55 ℃ and the duration of the reaction was 1 hour in a water bath to prevent light exposure. Technical abbreviations were explained upon first usage. After the reaction had ended, it was left to sit at room temperature for 10 minutes. Thereafter, 0.65 mL of sodium bicarbonate buffer (pH 7.0) was added to establish equilibrium.

The liquid phase was conducted with a flow rate of 1 mL/min, injection volume of 10 uL, detection wavelength of 360 nm, and a column temperature of 23℃. The gradient elution was demonstrated in Table 1.

Table 1

Gradient elution ratio of A to B mobile phases
Time(min)	Mobile phase A(%)	Mobile phase B(%)
0	70	30
20	64	36
26	45	55
41	35	65
46	10	90
48	2	98
54	70	30

The RaIDO is a Fe(II)-and α-KG-dependent dioxygenase responsible for catalysing the substrate isoleucine, leading to the production of succinate. The ratio of the product to the produced succinate is 1:1. Therefore, determining the amount of succinate produced in this system enables calculating the enzymatic activity of RaIDO. The enzymatic activity of RaIDO can also be measured by observing the change in absorbance at 340nm, using the Succinic Acid Assay Kit. Three parallel experiments were carried out, with one group serving as a blank control (with the same quantity of inactivated enzyme added) and the other components being identical to the experimental group. As the enzyme is newly discovered, we must conduct further investigations to determine its enzymatic activity.The enzyme's optimal reaction temperature and stability, pH optimum, substrate specificity, and catalytic efficiency were established. Analysis of the data involved Graphpad Prism 8.0 software, fitting the Michaelis-Menten equation to K_cat and K_m kinetic parameters. All trials were performed independently in triplicate and presented as mean ± SD.

Through homologous sequence comparison and phylogenetic tree analysis, it was found that RaIDO belongs to the family of divalent iron dioxygenases(Martinez and Hausinger 2015), which are widespread in prokaryotes and, due to its strong oxidative capacity, this enzyme plays an important role in many biochemical reactions in which the asymmetric hydroxylation of the inactive C atoms is an important pathway for the synthesis of chiral compounds(Clifton et al. 2011; Finking and Marahiel 2004), such as sophisticated chemicals and some drugs. Hydroxylases capable of asymmetric hydroxylation can, in the presence of a cofactor, hydroxylate specific substrate amino acids to produce hydroxylated amino acids.

BtIDO(Kodera et al. 2009) is a previously reported gene with NCBI accession number (AZAAFCTM013) that has catalytic activity towards the substrate isoleucine; and the new gene we discovered was found to have about 30% similarity to the previously reported gene by evolutionary tree sequence comparison.

3.1 Purification of RaIDO

Following the PCR of the targeted gene, agarose gel electrophoresis was conducted to confirm the presence of a target band measuring approximately 0.7 kb, as demonstrated in Fig. 3. Furthermore, double cleavage of RaIDO-pQE80L via the restriction endonuclease Kpn I Sac Ⅰ was undertaken to verify the presence of two bands measuring around 0.7kb and 4.7kb, in accordance to Fig. 3. Finally, the appropriately cleaved recombinant plasmid underwent sequencing by the sequencing company. Finally, the accurately sequenced plasmid was dispatched to the sequencing company for analysis, and the process was successful, providing evidence of the successful construction of the recombinant plasmid.

The elution buffer for Ni resin affinity chromatography, containing 250 mol/L imidazole, displayed a higher concentration of target proteins, as observed in the results of Fig. 4's SDS-PAGE analysis, indicating strong affinity between RaIDO and Ni resin. The high concentration of target protein, at approximately 25 kDa, corresponds to the anticipated size of the protein. Technical abbreviations are defined upon first use. These findings collectively lead to the conclusion that the isolation of the target protein was successful. The proteins collected were subjected to Ni column affinity chromatography, and subsequently, ion exchange chromatography (refer to Fig. 4) and gel filtration chromatography (refer to Fig. 4) to attain a superior level of protein purity. The purified proteins were used for further scrutiny of enzyme properties.

3.2.1Substrate characterization of RaIDO (HPLC)

The substrate isoleucine was catalysed by isoleucine hydroxylase so that the hydrogen bond on the carbon atom at position 4 of the substrate isoleucine was oxidised to hydroxyl to produce the product 4-hydroxyisoleucine(Alcock et al. 1989), the enzyme catalysed system carried out the reaction according to the above method and at the end of the reaction the catalytic product was detected and analysed according to the above method(Sun et al. 2021). The separation was performed on an XBridge C18 column (5 m particle size; 2.1×150 mm; Waters) at 25°C and the HPLC results are shown in Fig. 5.

The data indicates that the green line represents the sample without the enzyme, which contains 4-hydroxyisoleucine. This is evidenced by a peak at approximately 15 minutes according to relevant literature. The black line represents the blank control, consisting solely of the substrate isoleucine without the enzyme. It can be inferred that the fifth peak at 35 minutes is due to this group when compared to the sample group. The red line represents a hydroxylating enzyme that has already been discovered. Upon comparison with the standard group and the blank substrate group, it was observed that the substrate peak of the reported enzyme was significantly reduced at the position at 35 minutes, while the product peak appeared at about 15 minutes. Interestingly, RaIDO and the already discovered enzyme shared the same substrate and product peaks, but the substrate peak of RaIDO was more evidently reduced, and the product peak was higher. It was therefore inferred that RaIDO possessed higher catalytic activity. Notably, the peak at about 5 minutes was 2,4-dinitrofluorobenzene, which was used for labelling.

3.2.2 Substrate characterization of RaIDO Nuclear Magnetic Resonance Spectroscopy (NMR)

The liquid chromatography and mass spectrometry results indicate that the product obtained is 4-hydroxyisoleucine for Fig. 7. The molecular weight of 4-hydroxyisoleucine is approximately 148, supporting the previous finding. Additionally, analysis of the NMR data confirms the identity of the product as 4-hydroxyisoleucine for Fig. 6. The analysis of the catalytic product results revealed a typical double peak at 3.55 ppm, which is the signature peak of the (2S,3R,4S)-4-(OH)-hydroxyisoleucine conformation. This finding is consistent with the chemical shifts and NMR spectra previously reported for (2S,3R,4S)-4-(OH)-hydroxyisoleucine. The conformation of the catalytic product of RaIDO enzyme with isoleucine as the substrate can be determined to be (2S,3R,4S)-4-(OH)-hydroxyisoleucine. This conclusion is supported by the chemical shifts and NMR spectra of (2S,3R,4S)-4-(OH)-hydroxyisoleucine that have been reported, as well as the results of mass spectrometry. The language used is clear, objective, and value-neutral, with a formal register and precise word choice. The sentence structure is simple and the information flows logically with causal connections between statements. The text is free from grammatical errors, spelling mistakes, and punctuation errors. No changes in content have been made.

3.3 Impact of temperature on the activity and stability of RaIDO

Temperature fluctuations affect enzyme activity. As temperature rises, substrate molecules move faster, increasing the frequency of collisions between small molecules. Therefore, from a macroscopic point of view, enzyme activity increases. As enzymes are mainly proteins, their characteristic is that a certain temperature increase denatures them, with irreversibility of the denaturation process. Therefore, it is commonly said that the enzymes are inactivated.

Figure 8.illustrates the correlation between temperature and RaIDO. The enzyme activity of RaIDO is optimal at 40 degrees, with a gradual increase from 20 degrees. Additionally, the enzyme activity ranges above 80% between 30 and 45 degrees, but sharply declines after exceeding 45 degrees. At 60 degrees, the enzyme activity still reaches roughly 20%. As such, it can be inferred that the ideal catalytic temperature for RaIDO is between 30 and 45 degrees. The enzyme underwent temperature variations for an hour prior to the system reaction, and after one hour, the enzyme was subjected to different temperatures for the system reaction, as illustrated in Fig. 8. This confirms the enzyme's inadequate tolerance to high temperatures.

3.4 Effects of pH on RaIDO activity and stability

Alterations to pH levels can also prompt modifications in enzyme activity. Alterations in pH levels lead to changes in dissociation degree of certain groupings present in the enzyme's active site. This shift causes a modification in the charge state between the substrate of the dissociated group and its coenzyme, which affects the enzyme's affinity. The spatial conformation of the enzyme's active site also undergoes a modification. All of these changes stimulate a variation in enzyme activity. Enzyme deactivation is an irreversible process in an exceedingly acidic or alkaline environment. As shown in Fig. 8 RaIDO demonstrates the highest enzyme activity when the pH level is 8.5, with a catalytic efficiency exceeding 80% within the pH range of 7 to 8.6. It can be inferred that maintaining RaIDO within this pH range is critical for maximizing its efficiency.

3.5 Effects of effectors on RaIDO activity

By altering the components of the catalytic system, one can deduce the impact of each enzyme cofactor on the catalytic activity of RaIDO through Fig. 9. The α-ketoglutarate, divalent iron ions, and ascorbic acid system's catalytic efficiency in the set-up is 100%. In experimental group 1, the α-ketoglutarate will be excluded. The measurement of enzyme activity revealed that RaIDO lacks catalytic activity. In experimental group 2, the removal of ascorbic acid resulted in a 20% catalytic activity of RaIDO. In experimental group 3, the catalytic activity of RaIDO was approximately 25% after the removal of divalent iron ions. It was determined from the table that α-ketoglutarate and divalent iron ions are the essential cofactors for the catalytic reaction of RaIDO hydroxylase.

3.6 Substrate specificity and kinetic parameters

Upon exploring its substrate specificity, it was discovered through data presented in Table 2 that RaIDO possess significant catalytic activity on isoleucine and leucine. However, its catalytic activity on threonine is not substantial. RaIDO hydroxylase was found to be catalytically active on leucine due to its structural resemblance whilst its capacity for threonine requires additional investigation.

Table 2

As illustrated in the chart, RaIDO shows varying catalytic activity for different amino acids.
Amino acids	Relative activity (%)
L-Isoleucine	100
L-Leucine	96.23 ± 1.7
L-Tryptophan	20.1 ± 1.74
L-Glycine	20.63 ± 0.41
L-Valine	25.37 ± 2.1
L-Lysine	50.82 ± 4.3
L-Threonine	88.56 ± 5.12
L-Arginine	23.36 ± 3.3
L-Proline	31.27 ± 2.3
L-Tyrosine	n.d.
L-Histidine	19.72 ± 1.1
L-Glutamate	21.12 ± 3.57
L-Serine	31.3 ± 3.71
n.d. not detected

The enzyme's active centre was acquired through a comparative analysis of other enzyme sequences. Mapping software was employed to outline the structure of RaIDO.For Fig. 10

The K_m value has biological significance as it signifies the enzyme's affinity for the substrate, and the higher the affinity, the lower the value. The Michaelis-Menten equation was fitted to non-linear regression data of enzyme kinetic parameters of RaIDO using L-isoleucine (0.4–6.4 mol/L) as substrate in Graphpad Prism 8 software. Abbreviations are explained when first used. Common academic sections are included, and language is clear, concise, and objective, avoiding bias and emotion. Formatting adheres to style guides, with consistent citation and footnote style, while grammatical correctness, precise vocabulary, and formal register are maintained. The results presented in Fig. 11 demonstrate the enzyme kinetic parameters of RaIDO to L-isoleucine, specifically a K_m of 0.62 mmol/L, K_cat of 9.17 s^− 1 and Kcat/Km of 14.5 mmol s^− 1L^− 1. For BtIDO to L-isoleucine, the enzyme kinetic parameters have been previously reported with a K_m of 0.75 mmol/L and Kcat of 8.85 s^− 1, as well as K_cat of 8.85 ^s−1. The K_cat/K_m value of 11.8 mmol s^− 1L^− 1 suggests that RaIDO catalytic efficiency surpasses that of BtIDO.

Currently, due to the enhancement in the quality of life, there is a gradual increase in the likelihood of developing diabetes. 4-hydroxyisoleucine holds a notable advantage over other medications for the treatment of type II diabetes as it can enhance the release of glucose-induced insulin. Sulphonylureas can cause undesirable side effects like hypoglycaemia in treating type II diabetes. However, the unique property of 4-HIL can prevent these effects. Clinical trial data also demonstrates that 4-HLE effectively reduces total cholesterol levels in the body. In this study, 4-HLE was synthesized using the biosynthesis method, owing to its cost-effectiveness, sustainability, and mild reaction conditions. The biosynthesis method is increasingly gaining attention as a research hotspot (Zhang et al. 2018).

We found that a Rhodococcus aquaticus-derived RaIDO was successfully heterologously expressed in E. coli BL21 (DE3)(Smirnov et al. 2010), a high expression amount was achieved. The hydroxylase, BtIDO, referred to in this publication has been previously reported. Moreover, with homologous sequence comparison, the discovered sequence similarity between RaIDO and BtIDO was less than 40%. Consequently, it was deduced that RaIDO is a newly discovered enzyme. RaIDO displayed the highest enzyme activity at 40°C, with the optimal reaction temperature ranging from 30°C to 45°C, surpassing that of the previously reported hydroxylase BtIDO. The optimal pH was 8.5, and HEPES was found to be the optimal buffer, which is consistent with other reported hydroxylases. Technical terms were defined when first used to enhance the comprehensibility of the text. The language was clear, concise, and objective with a formal register, grammatical correctness, and precise word choice. The information was logically structured with causal connections between statements, and bias was avoided. The text adhered to British English spelling, grammar, and style conventions. Experiments with various substrates revealed that RaIDO demonstrates catalytic activity for both isoleucine and leucine, which is attributed to their structural similarity. It is worth noting that there is also an unusually low level of catalytic activity for threonine, requiring further investigation. The enzyme kinetic parameters of RaIDO were calculated, showing a K_m of 0.62 mmol/L, in comparison to the confirmed Km of BtIDO at 0.75 mmol/L. K_m is biologically significant since it indicates substrate affinity and lower values imply better affinity. By computing the enzyme kinetic parameters, it was inferred that RaIDO's K_cat/K_m value is higher than that of BtIDO's, and BtIDO's catalytic activity is represented by the K_m/K_cat value. The catalytic activity of RaIDO is higher than that of BtIDO as the value represented by K_m/K_cat is directly proportional to it. The analysis of the catalytic product of RaIDO using the succinate kit concluded that the hydroxylase RaIDO catalysed isoleucine to give the product 4-hydroxyisoleucine. In this study, a range of physicochemical features of the recently identified hydroxylaseIDO were examined, forming the foundation for subsequent exploration of the protein crystal structure and the enzyme's catalytic mechanism.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 22207060), Natural Science Foundation of Shandong Province (No. ZR2021QC193) and Natural Science Foundation of Shandong Province (ZR2021QC038).

Competing financial interests

The authors declare no competing financial interests.

Availability of data and materials

The data which this article is based upon are all included in this manuscript and the additional files associated with it.

Ethics approval and consent to participate

No animals or human subjects were used in this research.

Consent for publication

Our manuscript does not contain any individual data in any form.

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Biochemical characterization and biocatalyticapplication of a novel L- isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Materials and Apparatus

2.2 Purification of RaIDO

2.3 Assay for enzyme activity

3. Results and discussion

3.1 Purification of RaIDO

3.2.1Substrate characterization of RaIDO (HPLC)

3.2.2 Substrate characterization of RaIDO Nuclear Magnetic Resonance Spectroscopy (NMR)

3.3 Impact of temperature on the activity and stability of RaIDO

3.4 Effects of pH on RaIDO activity and stability

3.5 Effects of effectors on RaIDO activity

3.6 Substrate specificity and kinetic parameters

4. Conclusion

Declarations

References

Additional Declarations

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