Protein structure modeling indicates hexahistidine‐tag interference with enzyme activity

Freydank, Anna-Carolin; Brandt, Wolfgang; Dräger, Birgit

doi:10.1002/prot.21905

Cited by 44 publications

(32 citation statements)

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“…The WT gyrA and gyrB genes were amplified from M. tuberculosis H37Rv (1, 2, 34). The full-length gyrA and gyrB genes were inserted downstream of the T7 promoter in expression vectors pET-20b (ϩ) and pET19b, respectively, for expression as His-tagged recombinant proteins since the His tag has been previously shown not to interfere with the catalytic functions of GyrA and GyrB (14). Resulting plasmids pTB-A (gyrA in pET-20b) and pTB-B (gyrB in pET-19b) were used to transform E. coli Rosetta-gami 2 (DE3)/pLysS and BL21(DE3)/pLysS, respectively.…”

Section: Resultsmentioning

confidence: 99%

Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance

Kim

Nakajima

Yokoyama

et al. 2011

Antimicrob Agents Chemother

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Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5-to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5-to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.A major human infectious disease, tuberculosis (TB) is estimated to affect approximately one-third of the world's population, and 95% of cases occur in developing countries (15,30,31). Current estimates show that approximately 9.4 million new cases and nearly 1.7 million deaths from TB occur each year, and TB remains a major cause of premature death (36).The increased incidence of multidrug-resistant (MDR) TB (TB resistant to more than two anti-TB drugs, including rifampin and isoniazid [35]) has hampered the treatment and control of TB and is associated with an increase in mortality rates in people with TB (3,37,40). Consequently, the required drug dosage for the treatment of TB has dramatically increased (38), and fluoroquinolones (FQs) are now considered to be important second-line anti-TB agents (13,20

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Section: Resultsmentioning

confidence: 99%

Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance

Kim

Nakajima

Yokoyama

et al. 2011

Antimicrob Agents Chemother

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“…His tags are commonly left on the expressed and purified proteins, presuming that the small size and charge of the tag will not alter the properties of the protein. The His tag has been shown to have no effect on the activity of some proteins (31,55), whereas the addition of a His tag to other proteins has been reported to alter enzyme activity (2,15,21) or dimerization state (56). The His tag is, however, a very useful tool for the detection and purification of hardto-express novel proteins.…”

Section: Discussionmentioning

confidence: 99%

NovelCoprinopsis cinereaPolyesterase That Hydrolyzes Cutin and Suberin

Kontkanen

Westerholm-Parvinen

Saloheimo

et al. 2009

Appl Environ Microbiol

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Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderma reesei under the control of an inducible cbh1 promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter ؊l purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C 2 to C 10 ) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.Cutin and suberin are lipid-derived insoluble polyesters that serve as structural components of the outer barriers of plants. Significant amounts of cutin are present in different agricultural and food raw materials, such as cereals, berries, fruits, and vegetables, and their processing by-products, whereas the bark of cork oak and birch is rich in suberin. These polymers are estimated to be the third most abundant natural polymers after cellulose and lignin (22). The weight of an isolated cuticle ranges from 0.45 to 0.8 mg cm Ϫ2 (leaf cuticles) to 2 mg cm Ϫ2(fruit cuticles), of which 40 to 80% is formed by cutin (23).Cutin is primarily composed of ester-linked fatty acids, of which mono-, di-, and trihydroxy and epoxy fatty acids of the C 16 and C 18 families are the most abundant (25). Suberin consists of both a polyaliphatic fatty acid domain similar to that of cutin, with epoxy, hydroxy, and carboxylic acid functionalities, and a polyphenolic domain (4). The main suberin monomers are aliphatic long-chain ␣,-diacids and -hydroxyacids, together with glycerol (20). Cutin can be depolymerized by chemical cleavage of the ester bonds. However, epoxy, hydroxy, and carboxylic acid functionalities are not always retained when harsh chemical treatments are applied. Thus, enzymatic depolymerization of cutin is an attractive option and could result in formation of both monomers and oligomers with unmodified functionalities (26). Partial depolymerization of cork suberin by methanolysis has been found to form linear esters, glyceryl esters, and feruloyl esters, in which ferulic acid is linked to hydroxy acids (20).Cutinases and suberinases are polyesterases which are able to degrade or partially de...

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“…Purification by affinity chromatography using various fusion tags, cloned and expressed as a part of the protein of interest, often allows single-step column purification of the proteins: this technique may improve yields by increasing solubility or may positively affect the catalytic activity of the target protein [14]. On the other hand, His 6 -tag, as described here, allows simple immobilization of the enzyme on Ni-NTA columns and synthesis of the enzyme product strictosidine or its analogs in preparative amounts.…”

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confidence: 99%

Improved Expression of His₆‐Tagged Strictosidine Synthase cDNA for Chemo‐Enzymatic Alkaloid Diversification

Yang

Zou

Zhu

et al. 2010

Chemistry & Biodiversity

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Strictosidine synthase (STR1) catalyzes the stereoselective formation of 3alpha(S)-strictosidine from tryptamine and secologanin. Strictosidine is the key intermediate in the biosynthesis of 2,000 plant monoterpenoid indole alkaloids, and it is a key precursor of enzyme-mediated synthesis of alkaloids. An improved expression system is described which leads to optimized His(6)-STR1 synthesis in Escherichia coli. Optimal production of STR1 was achieved by determining the impact of co-expression of chaperones pG-Tf2 and pG-LJE8. The amount and activity of STR1 was doubled in the presence of chaperone pG-Tf2 alone. His(6)-STR1 immobilized on Ni-NTA can be used for enzymatic synthesis of strictosidines on a preparative scale. With the newly co-expressed His(6)-STR1, novel 3alpha(S)-12-azastrictosidine was obtained by enzymatic catalysis of 7-azatryptamine and secologanin. The results obtained are of significant importance for application to chemo-enzymatic approaches leading to diversification of alkaloids with novel improved structures.

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Protein structure modeling indicates hexahistidine‐tag interference with enzyme activity

Cited by 44 publications

References 50 publications

Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance

Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance

NovelCoprinopsis cinereaPolyesterase That Hydrolyzes Cutin and Suberin

Improved Expression of His₆‐Tagged Strictosidine Synthase cDNA for Chemo‐Enzymatic Alkaloid Diversification

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Protein structure modeling indicates hexahistidine‐tag interference with enzyme activity

Cited by 44 publications

References 50 publications

Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance

Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance

NovelCoprinopsis cinereaPolyesterase That Hydrolyzes Cutin and Suberin

Improved Expression of His6‐Tagged Strictosidine Synthase cDNA for Chemo‐Enzymatic Alkaloid Diversification

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Product

Resources

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Improved Expression of His₆‐Tagged Strictosidine Synthase cDNA for Chemo‐Enzymatic Alkaloid Diversification