Biotransformation of β-ionone by engineered cytochrome P450 BM-3

Wild-type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has a low hydroxylation activity for β-ionone (<1 min⁻¹). Substitution of phenylalanine by valine at position 87 led to a more than 100-fold increase in β-ionone hydroxylation activity (115 min⁻¹). Enzyme activity could be further increased by both site-directed and random mutagenesis. The mutant R47L Y51F F87V, designed by site-directed mutagenesis, and the mutant A74E F87V P386S, obtained after two rounds of error-prone polymerase chain reaction, exhibited an increase in activity of up to 300-fold compared to the wild-type enzyme. The triple mutant R47 LY51F F87V exhibited moderate enantioselectivity, forming (R)-4-hydroxy-β-ionone with an optical purity of 39%. All mutants regioselectively converted β-ionone into 4-hydroxy-β-ionone. The regioselectivity is determined amongst others by the absolute configuration of the substrate.     

Regioselectivity-driven evolution of CYP102D1 for improved synthesis of 3′-ortho-dihydroxyisoflavone

Daidzein is a major component of isoflavones, and its hydroxylated forms are valuable phytochemicals with anti-cancer and anti-oxidant activity. Due to the limitations of chemical synthesis of these hydroxylated structures, alternative enzymatic synthesis has been attempted. Previously, several protein-engineering approaches using CYP102D1 were investigated; these produced mutants with daidzein hydroxylation activity and regioselectivity through rational design (F96V/M246I) and saturation mutagenesis (A273H/G274E/T277G). However, the generated mutants have low regioselectivity (F96V/M246I) or low hydroxylation activity (A273H/G274E/T277G). Here, we characterized mutants capable of catalyzing C3′-specific daidzein hydroxylation with enhanced hydroxylation activity and regioselectivity. In order to obtain regioselectivity toward the daidzein C3′-position, site-saturation mutagenesis on the substrate-binding region of CYP102D1 F96V/M246I was investigated. A high-throughput screening assay was then performed, based on O-dealkylation activity against the daidzein analog substrate 4′-O-methyl-daidzein. This resulted in a mutant with more than 23-fold improved hydroxylation activity (55.6 ± 17.9 μM⁻¹ min⁻¹, or 48.4 mg/L titer) and regioselectivity over the 3′/6-position that was increased by three-fold (from 0.9 to 2.6) compared with the F96V/M246I template enzyme. Furthermore, we carried out docking simulation studies that could partially explain the effects of these mutations on C3′-specific hydroxylation activity.     

Site-directed mutagenesis of cysteine residues of <Emphasis Type="Italic">Luciola mingrelica</Emphasis> firefly luciferase

Single mutants (C62S, C62V, C86S, C146S, C164S), double mutants (C62/146S, C62/164S, C86/146S, C146/164S), and triple mutant C62/146/164S of the Luciola mingrelica firefly luciferase carrying C-terminal His6-tag were obtained on the basis of plasmid pETL7 by site-directed mutagenesis. Bioluminescence and fluorescence spectra were not altered by the introduced mutations. In the case of mutants C86S, C86/146S, C62/164S, and the triple mutant C62/146/164S, the K _m^ATP and K_m^LH₂ K_m^{LH_2 } values were increased by a factor of ∼1.5–1.9. Their expression level, specific activity, and thermal stability were significantly decreased. The other mutations had almost no effect on the K _m^ATP and K_m^LH₂ K_m^{LH_2 } values, specific activity, and thermal stability of the enzyme. Thermal stability of the C146S mutant was increased by a factor of ∼2 and 1.3 at 37 and 42°C, respectively. The possible mechanism of the influence of these mutations on properties and structure of the enzyme is discussed.     

Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> polyhydroxyalkanoate synthase

The F420S substitution enhances the specific activity of Ralstonia eutropha PHA synthase (PhaC_Re). We have now carried out site-directed saturation mutagenesis of F420 of PhaC_Re and, amongst the F420 mutants, the F420S mutant gave the highest poly(3-hydroxybutyrate) (PHB) content. In vitro activity assay showed that the F420S enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. Enhancement of PHB accumulation was achieved by combination of the F420S mutation with a G4D mutation, which conferred high PHB content and high in vivo concentration of PhaC_Re enzyme. The G4D/F420S mutant gave a higher PHB content and in vivo concentration of PhaC_Re enzyme than the F420S mutant, while the molecular weight of the PHB polymer of the double mutant was similar to that of the F420S mutant.     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.     

Cumulative effect of amino acid replacements results in enhanced thermostability of potato type L alpha-glucan phosphorylase

The thermostability of potato type L alpha-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations-Phe39-->Leu (F39L), Asn135-->Ser (N135S), and Thr706-->Ile (T706I)-by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60 degrees C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65 degrees C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.     

Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards gamma- and delta-positions

Cytochrome P450 BM-3 monooxygenase from Bacillus megaterium (CYP102A1) catalyzes the subterminal hydroxylation of fatty acids with a chain length of 12-22 carbons. Wild-type P450 BM-3 oxidizes saturated fatty acids at subterminal positions producing a mixture of omega-1, omega-2 and omega-3 hydroxylated products. Using a rational site-directed mutagenesis approach, three new elements have been introduced into the substrate binding pocket of the monooxygenase, which greatly changed the product pattern of lauric acid hydroxylation. Particularly, substitutions at positions S72, V78 and I263 had an effect on the enzyme regioselectivity. The P450 BM-3 mutants V78A F87A I263G and S72Y V78A F87A were able to oxidize lauric acid not only at delta-position (14% and 16%, respectively), but also produced gamma- and beta-hydroxylated products. delta-Hydroxy lauric and gamma-hydroxy lauric acid are important synthons for the production of the corresponding lactones.     

Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The stability and specific activity of endo-β-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4–8.8) and thermotolerance (entirely active at 55°C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4–5.2; thermostability, inactive at 55°C for 30 min). k _cat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K _m became twice of that of the wild type.     

Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase

The thermostability of potato type L α-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39→Leu (F39L), Asn135→Ser (N135S), and Thr706→Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.     

Semi-rational site-directed mutagenesis of phyI1s from <Emphasis Type="Italic">Aspergillus niger</Emphasis> 113 at two residue to improve its phytase activity

Through alignment of amino acid sequences among different phytases, we found that the amino acid at residues 53 and 91 vary broadly. To prove that the amino acid at residues 53 and 91 were related to phytase specific activity, two single mutant phyI1s Q53R and K91D were obtained by site-directed mutagenesis strategy. None of the single amino acid residues in the two mutants was in a position reported to be important for catalysis or substrate binding. Kinetic analysis of the phytase activity of the two mutants (Q53R and K91D) indicated that the mutants were attributed to 2.2- and 1.5-fold increased specific activity, and a 1.47- and 1.16-fold increased affinity for sodium phytate. In addition, the overall catalytic efficiency (k _cat/K _m) of the two mutants was improved 4.08- and 2.84-fold compared to that of the wild type. Such mutants will be instrumental for the structure–function study of the enzyme and for industrial application.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcusalbus

The cellobiose 2-epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4-O-β-d-glucopyranosyl-d-mannose and 4-O-β-d-galactopyranosyl-d-mannose (epilactose). Based on the sequence alignment with N-acetyl-d-glucosamine 2-epimerases of known structure and on a homology-modeled structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. We found that R52, H243, E246, W249, W304, E308, and H374 were absolutely required for the activity of RaCE. F114 and W303 also contributed to catalysis. These residues protruded into the active-site cleft in the model (α/α)₆ core barrel structure.     

Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues

Previous reports have shown that a unique bacterial dye-decolorizing peroxidase from the cyanobacterium Anabaena sp. strain PCC7120 (AnaPX) efficiently oxidizes both recalcitrant anthraquinone dyes (AQ) and typical aromatic peroxidase substrates. In this study, site-directed mutagenesis to replace five Met residues in AnaPX with high redox residues Ile, Leu, or Phe was performed for the improvement of the enzyme stability toward H₂O₂. The heme cavity mutants M401L, M401I, M401F, and M451I had significantly increased H₂O₂ stabilities of 2.4-, 3.7-, 8.2-, and 5.2-fold, respectively. Surprisingly, the M401F and M451I retained 16% and 5% activity at 100 mM H₂O₂, respectively, in addition to maintaining high dye-decolorization activity toward AQ and azo dyes at 5 mM H₂O₂ and showing a slower rate of heme degradation than the wildtype enzyme. The observed stabilization of AnaPX may be attributed to the replacement of potentially oxidizable Met residues either increasing the local stability of the heme pocket or limiting of the self-inactivation electron transfer pathways due to the above mutations. The increased stability of AnaPX variants coupled with the broad substrate specificity can be potentially useful for the further practical application of these enzymes especially in bioremediation of wastewater contaminated with recalcitrant AQ.     

Identifying functional residues in Arabidopsis thaliana zeta class glutathione S-transferase through screening inactive point mutants

The functional residues of z-class glutathione S-transferase were identified by screening inactive point mutants from a random mutagenesis library. First, a random mutant library was constructed using error-prone polymerase chain reaction, and then candidate inactive mutants were screened by a high-throughput colorimetric assay. Twenty-five mutants were obtained, and 12 that formed inclusion bodies were discarded. The remaining 13 mutants that expressed soluble protein were used for accurate quantification of enzymatic activity and sequencing. The mutants W15R, C19Y, R22H/K83E, P61S, S73P, S109P, and Q112R were found to have activity lower than 1% of the wild-type and were considered as “inactive mutants”, whereas the mutants K83E, Q102R, and L147F still have a large fraction of the activity and were thus considered as “partially inactivated mutants”. Molecular modeling experiments disclosed that mutations resulting in inactivation of the enzyme were found in or near the binding pocket, whereas mutations resulting in partial inactivation were distant from both substrates. The role of the residue Ser73 in the enzyme was verified by site-directed mutagenesis. The result suggested that screening inactive point mutants from a random mutagenesis library is an efficient way of identifying functional residues in enzymes.     

The Role of β20–β21 Loop Structure in Insecticidal Activity of Cry1Ac Toxin from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The β20–β21 loop is a unique structure in the domain III of Bacillus thuringiensis Cry proteins. In this study, the role of β20–β21 loop on insecticidal activity of Cry1Ac toxin was investigated. 10 residues in β20–β21 loop were substituted with alanine using PCR-based site-directed mutagenesis. All mutants were capable of producing diamond-shaped crystal and expressing a protein sized 130 kDa. The mutants S581A and I585A enhanced toxicity against Helicoverpa armigera larvae dramatically, while most of the rest mutants possess a reduced toxicity at different degrees. Indoor bioassay result revealed that mutants S581A and I585A had a 1.72- and 1.89-fold increasing in toxicity against Helicoverpa armigera larvae compared with the wild-type strain, respectively; On the contrary, G583A experienced a significant reduced insecticidal activity. Three-dimensional analysis of Cry1Ac5 protein demonstrated that the side chain of residues T579, S580, L582, and I585 extended to the surface of the protein, and might participate in the interaction between the protein and its receptor, whereas side chain of residues N576, F578, S581, N584, and V586 preferred the inside of the protein, and which might be critical to the stability of the protein structure. Our study for the first time clarified the special properties and the functions of the β20–β21 loop in domain III of Cry1Ac5. These findings also provided the latest biological evidence for the recognition and binding mechanism of the domain III in Cry1Ac, and its role in maintaining the structure stability of Cry1Ac.     

Active site analysis of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase from <Emphasis Type="Italic">Nocardia tartaricans</Emphasis> using homology modeling and site-directed mutagenesis

Cis-epoxysuccinate hydrolase (CESH, EC 3.3.2.3) from Nocardia tartaricans is known to catalyze the opening of an epoxide ring of cis-epoxysuccinate (CES), thereby converting it to corresponding vicinal diol, l(+)-tartaric acid. An attempt has been made to build a 3D homology model of CESH to investigate the structure–function relationship, and also to understand the mechanism of the enzymatic reaction. Using a combination of molecular-docking simulation and multiple sequence alignment, a set of putative residues that are involved in the CESH catalysis has been identified. Functional roles of these putative active-site residues were further evaluated by site-directed mutagenesis. Interestingly, the mutants D18A, D18E, Q20E, T22A, R55E, N134D, K164A, H190A, H190N, H190Q, D193A, and D193E resulted in complete loss of activity, whereas the mutants Y58F, T133A, S189A, and Y192D retained partial enzyme activity. Furthermore, the active-site residues responsible for the opening of CES were analyzed, and the mechanism underlying the catalytic triad involved in l(+)-tartaric acid biosynthesis was proposed.     

Identification of glutamate residues important for catalytic activity of Bacillus stearothermophilus leucine aminopeptidase II

Each of four conserved glutamate residues of Bacillus stearothermophilus leucine aminopeptidase II (BsLAPII) was replaced with aspartate, lysine, and leucine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes were purified to homogeneity by nickel-chelate chromatography and the molecular mass of the subunit was determined to be 44.5 kDa by SDS-PAGE. The specific activity for the Glu-316 and Glu-340 mutants was completely abolished, while Glu-249 mutants showed comparable activity to that of the wild-type BsLAPII. Compared with the wild-type enzyme, the E250D and E250L mutant enzymes retained less than 18% of the enzyme activity and exhibited a dramatic decrease in the value of k _cat/K _m. These observations indicate that Glu-250, Glu-316, and Glu-340 residues are critical for the catalytic activity of BsLAPII.     

Hydrophobic residues in small ankyrin 1 participate in binding to obscurin

Abstract

Small ankyrin-1 is a splice variant of the ANK1 gene that binds to obscurin A. Previous studies have identified electrostatic interactions that contribute to this interaction. In addition, molecular dynamics (MD) simulations predict four hydrophobic residues in a ‘hot spot’ on the surface of the ankyrin-like repeats of sAnk1, near the charged residues involved in binding. We used site-directed mutagenesis, blot overlays and surface plasmon resonance assays to study the contribution of the hydrophobic residues, V70, F71, I102 and I103, to two different 30-mers of obscurin that bind sAnk1, Obsc_6316–6345 and Obsc_6231–6260. Alanine mutations of each of the hydrophobic residues disrupted binding to the high affinity binding site, Obsc_6316–6345. In contrast, V70A and I102A mutations had no effect on binding to the lower affinity site, Obsc_6231–6260. Alanine mutagenesis of the five hydrophobic residues present in Obsc_6316–6345 showed that V6328, I6332, and V6334 were critical to sAnk1 binding. Individual alanine mutants of the six hydrophobic residues of Obsc_6231–6260 had no effect on binding to sAnk1, although a triple alanine mutant of residues V6233/I6234/I6235 decreased binding. We also examined a model of the Obsc_6316–6345-sAnk1 complex in MD simulations and found I102 of sAnk1 to be within 2.2Å of V6334 of Obsc_6316–6345. In contrast to the I102A mutation, mutating I102 of sAnk1 to other hydrophobic amino acids such as phenylalanine or leucine did not disrupt binding to obscurin. Our results suggest that hydrophobic interactions contribute to the higher affinity of Obsc_6316–6345 for sAnk1 and to the dominant role exhibited by this sequence in binding.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.