MINIREVIEW Cholesterol oxidase: biotechnological applications Loredano Pollegioni, Luciano Piubelli and Gianluca Molla Dipartimento di Biotecnologie e Scienze Molecolari, Universita ` degli studi dell’Insubria, Varese, Italy, and Centro di Ricerca Interuniversitario in Biotecnologie Proteiche ‘‘The Protein Factory’’, Politecnico di Milano and Universita ` degli studi dell’Insubria Introduction In eukaryotes, cholesterol is essential for maintaining cell membrane structure and for synthesizing a number of important compounds. Moreover, improper mainte- nance of cholesterol concentrations can severely affect the physiological function of an organism. Cho- lesterol oxidase (ChOx, EC 1.1.3.6), or more precisely, 3b-hydroxysterol oxidase, is a bacterial enzyme that has proven to be very useful in biotechnological appli- cations related to the detection (and conversion) of cholesterol and to the disruption of cholesterol-con- taining membranes. As described in the two accompa- nying reviews in this miniseries [1,2], ChOx catalyzes the oxidation of the C3-OH group of cholesterol (and other sterols) to give the corresponding D 5 -3-ketone (cholest-5-en-3-one) and its isomerization to D 4 -3-ke- tone (cholest-4-en-3-one, see Scheme 1 in the first mini- review [1]). In bacteria, ChOx is the first enzyme in the catalytic pathway that yields propionate and acetate as final products. Importantly, there is no mammalian homolog of ChOx. Since 1973, ChOx has been used in clinical chemistry to measure serum cholesterol. ChOx research has grown largely in the past several years: a PubMed search for the term ‘cholesterol oxidase’ yielded 124, 212 and > 220 papers in the periods 1980–1989, 1990– 1999 and 2000–2008, respectively. ChOx is produced by two types of bacteria: (a) nonpathogenic bacteria, which utilize cholesterol as a carbon source; and (b) Keywords biocatalysis; cholesterol; cholesterol determination; diagnostic enzyme; enzyme biotechnology; flavoproteins; virulence Correspondence Loredano Pollegioni, Dipartimento di Biotecnologie e Scienze Molecolari, Universita ` degli studi dell’Insubria, via J.H. Dunant 3, 21100 Varese, Italy Fax: 0332 421500 Tel: 0332 421506 E-mail: loredano.pollegioni@uninsubria.it (Received 23 July 2009, revised 2 September 2009, accepted 10 September 2009) doi:10.1111/j.1742-4658.2009.07379.x Cholesterol oxidase is a bacterial FAD-containing flavooxidase that catalyzes the first reaction in cholesterol catabolism. Indeed, this enzyme catalyzes two reactions: the oxidation of the C 3 -OH group of cholesterol (and other sterols) to give cholest-5-en-3-one; and its isomerization to cholest-4-en-3-one. In the past several years, the structural and functional characterization of choles- terol oxidase has been developed together with its application as a biological tool. Cholesterol oxidase has been used in biocatalysis for the production of a number of steroids, as an insecticidal protein against boll weevil larvae and, in particular, as a diagnostic enzyme for determining serum levels of choles- terol. These applications prompted various laboratories worldwide to isolate this flavooxidase from different sources and to improve its properties by pro- tein engineering, further increasing our knowledge on its structure–function relationships. These studies also discovered new physiological roles for cho- lesterol oxidase (e.g. in virulence and as an antifungal sensor). We assume that the investigations of cholesterol oxidase and its applications will con- tinue to grow quickly in the near future, in particular to uncover unexpected, new areas of application. Abbreviations BsChOx, Brevibacterium sterolicum cholesterol oxidase; ChOx, cholesterol oxidase (EC 1.1.3.6); ReChOx, Rhodococcus equi cholesterol oxidase; ShChOx, Streptomyces hygroscopicus cholesterol oxidase. FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS 6857 pathogenic bacteria, which require ChOx for infection of the host macrophage because of its ability to alter the physical structure of the lipid membrane by con- verting cholesterol into cholest-4-en-3-one (see below). Both pathogenic and nonpathogenic bacteria upregu- late the expression of ChOx in the presence of choles- terol. This review summarizes our knowledge concerning the production of ChOx and its main uses. By under- standing the structure–function relationships of this flavoenzyme and the mode of action by which it alters the physical properties of lipid membranes, together with its overexpression in recombinant systems, we may discover new and ⁄ or optimized applications of ChOx. Sources of ChOx A list description of ChOx-containing microorganisms was published in 2000 [3]. This enzymatic activity is produced by a variety of microorganisms that are found in different environments: a detailed list is reported in the Supplementary materials section. Microorganisms such as Arthrobacter, Rhodococcus equi, Nocardia erythropolis, Mycobacterium and Nocardia rhodochrous produce an intracellular ⁄ membrane ChOx, while Pseudomonas, Schizophyllum commune, Brevibacteri- um sterolicum, Streptoverticillum cholesterolicum and Streptomyces violascens produce an extracellular enzyme. As a general rule, ChOx enzymes require the prosthetic group FAD for their activity (both as a cova- lently or noncovalently bound cofactor). An exception to this could be the ChOx isolated from c-Proteobacteri- um Y-134, which was reported to contain covalently bound FMN as a cofactor [4]. This enzyme also differs in substrate specificity compared with other known ChOxs (e.g. it is not active on pregnenolone and ergosterol, see Table 1). Generally, the presence of cholesterol (or of a similar compound) in the growth medium induces the produc- tion of ChOx. Among the ChOx-producing bacteria, R. equi (previously identified as B. sterolicum ATCC 21387) [5] exhibits high levels of ChOx activity, but com- mercial use of this microorganism is limited because of its highly pathogenic nature (see below). This enzyme is the product of the gene choE and it is homologous to other secreted ChOxs identified in Streptomyces spp. (see Fig. S1). This protein also exhibits significant similarities to putative ChOxs encoded by Mycobacterium tubercu- losis and Mycobacterium leprae. Rhodococcus erythropo- lis ChOx was produced (using cholesterol solubilized in Tween-80 in the culture medium) both as an intracellular enzyme and an extracellular enzyme: the maximal production of both forms of ChOx was obtained after 70 h of growth, using a spray-dry method of preparation of cholesterol (up to 365 UÆL )1 and 1.7 UÆg )1 of cells) [6]. Rhodococcus (Nocardia) erythropolis IMET 7185 also produces ChOx: the addition of 1 gÆL )1 of cholesterol results in a fivefold increase in ChOx production, up to 3.3 UÆg )1 of cells [7]. The soil-isolated strain Rhodococ- cus sp. GK1 produces ChOx both as membrane-bound and extracellular forms [8]. The synthesis of ChOx depends on the presence of either phytosterols or hex- anoate as the sole carbon source; under these conditions, the membrane-bound ChOx was produced up to 100 UÆg )1 of cells and 400 UÆL )1 [8]. By contrast, the synthesis of ChOx from Burkholderia cepacia (wrongly classified as Pseudomonas sp. strain ST-200 and corre- sponding to the choS gene, Fig. S1) is not induced by cholesterol and this bacterial strain produced only up to 13 UÆL )1 [9]. The highest production levels have been reported in the nonpathogenic Streptomyces sp., reach- ing up to  2500 UÆL )1 in fermentation broth in terms of purified protein [10] (for the sake of comparison, the activity values not measured at 25 °C have been cor- rected for the effect of temperature). Because of the moderate productivity of the various microorganisms used and the requirement of adding cholesterol to culture media to induce ⁄ enhance produc- tion, the cost of ChOx remained relatively high [6,8]. The yield of ChOx production was enhanced by alter- ing the composition of the growth medium and the physical parameters of culture. In S. commune, ChOx production depends on the amount of oleic acid in the culture broth but, interestingly, this applies to the level of insoluble oleic acid rather than to the soluble com- ponent adsorbed onto the cell [3]. A detailed investiga- tion of the growth conditions was also reported for R. equi no. 23 grown in a batch fermentor: by adjust- ing the pH (from 6.5 to 7.5), the temperature (from 39 to 37 °C) and the agitation speed (from 200 to 300 rpm) after 24 h of growth, maximal production of ChOx (340 UÆL )1 , corresponding to 11 UÆh )1 ÆL )1 ) was reached in 30 h of culture at an aeration rate of 5LÆmin )1 [11]. To overcome the difficulties related to overproduc- tion of this flavoenzyme in the original organism, ChOx genes from several sources have been cloned and expressed as recombinant proteins. However, most of these recombinant microorganisms produce ChOx at levels close to those of the original source. An exception to this is the expression of the gene encoding Rhodococcus equi cholesterol oxidase (ReChOx) in Streptomyces lividans that resulted in a yield of protein production  85-fold higher than that from the natural organism [12]. Its expression in Biotechnological applications of sterol oxidase L. Pollegioni et al. 6858 FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS S. lividans (and in Escherichia coli) was accomplished by using various deletions within the 5¢-flanking (non- coding) region. When the ReChOx protein was fused with the N-terminal part of the lacZ protein, large amounts of inactive enzyme were produced as ‘inclu- sion bodies’. In contrast, by using a gene with less than 256 bp at the 5¢-flanking region, enzyme could be efficiently produced in S. lividans, secreting  30000 UÆL )1 into the culture medium. Recently, ReChOx was also efficiently synthesized in E. coli under the control of the pT7lac promoter in the pET28 plasmid (up to 3.7 UÆmg )1 of protein was Table 1. Relative activity of ChOx from different sources. In all cases, activity on cholesterol (k cat value) was taken to be 100%. Substrate ReChOx a BsChOx b ShChOx b c-Proteobacterium Y-134 c Chromobacterium DS-1 d CHO-U CHO-A Cholesterol HO 100 100 100 (100) 100 (100) 100 100 100 7a-or7b-OH- Cholesterol HO HO – 99, 82 – – – – – 7-Keto- cholesterol HO O –61 – – – – – Ergosterol HO 051 – – <3 0 12 b-Sitosterol HO 19 65 – – 19 29 80 Pregnenolone HO O 22 90 70 (42) 70 (30) 0 0 95 Cholestanol HO 13 – 80 (46) 119 (300) – – 95 a Left column: 0.6% Triton X-100, pH 7.5 [70]; right column: 0.7% propan-2-ol, pH 7.5 [71]. b 100 mM potassium phosphate, pH 7.5, 0.4% Thesit (data in parenthesis were obtained in 500 mM potassium phosphate) [72,73]. c 100 mM potassium phosphate, pH 7.0, 0.05% Triton X-100, pH 7.5 [4,74]. d 50 mM sodium phosphate buffer, pH 7.0, 64 mM sodium cholate, 0.34% Triton X-100 [24]. L. Pollegioni et al. Biotechnological applications of sterol oxidase FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS 6859 produced in the crude extract and 2200 UÆL )1 was produced in the fermentation broth) [13,14], as well as in Chromobacterium DS-1 ChOx (1870 UÆL )1 was produced in the fermentation broth and 2.3 UÆmg )1 of protein was produced in the crude extract) [15]. The only data concerning the production of true B. steroli- cum ChOx (i.e. that containing the FAD cofactor covalently linked to the apoprotein moiety) were obtained for E. coli using the pET24 plasmid (up to 4UÆmg )1 of protein was produced in the crude extract and 12000 UÆL )1 was produced in the fermentation broth) [F. Volonte ` , L. Pollegioni, G. Molla, F. Mari- nelli and L. Piubelli, personal communication]. The Streptomyces ChOx gene was also overexpressed in S. lividans by subcloning the choP-choA operon into a multicopy shuttle vector, which produced a 70-fold greater amount of ChOx than the original organism (i.e. up to 4400 UÆL )1 in the fermentation broth) [16]. Interestingly, more than 90% of the protein produced is extracellular. The same ChOx was then produced in E. coli JM109 under the P8 promoter, reaching a specific activity in the crude extract of 9.7 UÆmg )1 of protein after 3 days of growth [17]. A detailed review of the methods for extracting and purifying ChOx has been published previously [3]. ChOx as a biological tool Effect of detergents and solvents on ChOx activity and stability ChOx is an enzyme that interacts with membranes and micelles and acts on hydrophobic substrates whose sol- ubility is increased by using detergents and ⁄ or solvents. These compounds also affect the activity ⁄ stability of the flavoprotein. One of the first investigations on the effect of detergents on ReChOx activity showed a sig- moidal behavior versus cholesterol concentration at a Triton X-100 concentration of ‡ 0.3% (the corre- sponding Hill plot gave an n-value of 4) with a con- comitant increase in the K m value [18]. The transition from a simple Michaelis–Menten behaviour to a sig- moidal behavior was also recently observed for the V121A mutant of Streptomyces hygroscopicus choles- terol oxidase (ShChOx) at a fixed Triton X-100 con- centration of 0.077% (the activity decreases with an increase in Triton X-100 concentration) [19]. Val121 in ShChOx is located in a hydrophobic loop near the active site, which should play an important role in extracting cholesterol from micelles: the V121A substi- tution negatively affects this step only if cholesterol is solubilized using nonionic detergents (no sigmoidal behavior is observed when cholesterol is solubilized in propan-2-ol). For example, the enzymatic activity of Nocardia ChOx is induced upon the formation of mixed micelles of cholesterol with molecules of the detergent Surfal: the enzyme is half activated at a detergent concentration corresponding to its critical micelle concentration [20]. In 1988, Cees Veeger’s group published a very important work regarding clarification of the effect of the solvent ⁄ detergent on the reaction catalyzed by ChOx [21]. The dependence of catalytic activity of No- cardia ChOx on the composition of a solution com- posed of n-hexane, propan-2-ol and water (a detergentless microemulsion) revealed two maxima. The maximal catalytic activity in ternary systems is equal to that determined in aqueous solution, whereas the K m value increases with solvent concentration because of a preferential interaction with the surround- ing organic solvent with respect to the hydrophobic ChOx active site. By contrast, the stability profile shows a single sharp maximum corresponding to the microemulsion region of the phase diagram [21]; in any case, the storage stability is higher in aqueous solution. We have also systematically studied the effect of the nonionic detergents Thesit and Triton X-100, and of propan-2-ol (used as a substrate solubilizer), on ShChOx and Brevibacterium sterolicum cholesterol oxi- dase (BsChOx) activity [22]. At a low concentration of Thesit, activity increases for both enzymes, whereas at higher detergent concentrations (‡ 2–5%) the opposite effect occurs. On the other hand, Triton X-100 inacti- vates both enzymes at all concentrations. We thus deduced that these surfactants exert their effect by interacting with the enzymes and not by affecting micellar phenomena. Analogously, the increase in con- centrations of propan-2-ol (or other organic solvents) up to  5–10% (v ⁄ v) induces an increase in the activ- ity of both ShChOx and BsChOx and a decrease at higher solvent concentration. A significant difference between the two ChOx enzymes emerges when stability is analyzed as a function of concentration of propan-2- ol: BsChOx is rapidly inactivated, whereas for ShChOx 70% of the initial activity still remains after 5 h in the presence of 30% propan-2-ol [22]. This observation is in line with the stability to detergents reported in Fig. 1 (see below). The stability of ChOxs from different sources, in the presence of several detergents, was also compared after 1 h of incubation at 30, 45 and 60 °C [4,23,24]. The sta- bility at 30 °C was similar among the ChOxs analyzed (the only exception was observed in the presence of ‡ 0.1% SDS that fully inactivated all enzymes except for those from Burkholderia and Chromobacterium DS-1), with the most significant changes being observed Biotechnological applications of sterol oxidase L. Pollegioni et al. 6860 FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS after 1 h of incubation at 60 °C in the presence of nonionic detergents (see Fig. 1, which reports additional data from our experiments on covalent BsChOx and its noncovalent variant H69A mutant). At 0.5% Triton X-100, all ChOxs assayed showed a high stability, while in the presence of sodium cholate, only the ChOxs from Proteobacterium Y-134, Chromobacterium DS-1 and Pseudomonas were stable. Interestingly, the ChOxs from Proteobacterium Y-134 and Chromobacterium DS-1 were also more stable to temperature than all other ChOxs: they maintained more than 90% of the original activity after 1 h of incubation at 60 °C. The results obtained using BsChOx were significantly different from those observed with other ChOxs. Both wild-type and H69A BsChOxs showed very low stability under the conditions tested: the residual activity after 60 min of incubation at 60 °C was £ 3% in all cases, despite the presence of the covalent FAD linkage in the wild-type BsChOx. Analogously, a systematic comparison was carried out to determine the stability of ChOxs, from different sources, after 12–24 h of exposure to solvents at 37 °C [23,24]. Interestingly, only the enzyme from Burkholderia is not inactivated by 33% (v ⁄ v) acetone and propan-2-ol. Uses in biocatalysis Many Actinobacteria are highly efficient in oxidizing the 3-OH group of D 5 -hydroxysteroids coupled to D 5 fi D 4 isomerization, thus providing valuable inter- mediates for industrial steroid drug production. For example, R. equi DSM 89-133 was used to convert cho- lesterol and other sterols to androst-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione [25]: up to 82% of cholesterol was converted when the growth medium was supplemented with acetate. The synthesis of 7b-hy- droxytestosterone was achieved by incubating a C 8 -ester at the C-17 position of 5-androstene-3b,7b,17b-triol-17- caprylate with ChOx and subsequently eliminating the intermediate ester with porcine lipase [26]. Analogously, in the bioconversion of cholesterol into bile acids, ReChOx was used to convert 3b,7a -cholest-5-ene-3,7- diol to 7a-hydroxycholest-4-en-3-one, obtaining a yield higher than 90% [27]. As another alternative, ShChOx was used to prepare unsaturated C21 triols as reference standards to study adrenal steroid production in Smith-Lemli-Opitz syndrome, with the ultimate aim of developing a prenatal or a postnatal diagnostic method [28]. R. erythropolis ChOx was employed for the preparative oxidation of cyclic allylic, bicyclic (e.g. 10b-methyl-D 1(9) -2b-octalol) and tricyclic alcohols, as well as of a synthon to synthesize several ergot alkaloids [29]. This work also demonstrated a lack of enantiospec- ificity for the steroids. Recently, ChOx from Chromo- bacterium DS-1 was reported to catalyze the oxidation of cholesterol to hydroperoxy-cholest-4-en-3-one with the consumption of 2 mol of O 2 [24]. A B C Fig. 1. Comparison of the stability of ChOxs, from different sources, in the absence (A) and in the presence of 0.1% (B) and 0.5% (C) Triton X-100 (black bars) or sodium cholate (white bars). The enzyme (0.1 UÆmL )1 ) was incubated at 60 °C for 60 min in 100 m M potassium phosphate, pH 7.0, before the enzymatic activ- ity assay was performed: the residual activity was calculated as a percentage of the enzyme activity without heating and incubation. Data for Pseudomonas (Pse), Nocardia (Noc), Rhodococcus (Rho), Streptomyces (Str) and c-Proteobacterium Y-134 (Y-134) are from Isobe et al. [4] and for Chromobacterium sp. DS-1 are from Doukyu et al. [24]. Bre, BsChOx. n.d., not determined. L. Pollegioni et al. Biotechnological applications of sterol oxidase FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS 6861 With the ultimate goal of modifying the relative ratios of specific plant sterols to stanols [i.e. the hydro- genated forms having the C-5 double bond reduced (and which attracted attention because of their benefi- cial effect in reducing serum and low-density lipopro- tein cholesterol levels)], it is noteworthy that ShChOx was used for engineering oil seeds from rapeseed and soybean [30]. Clinical uses ChOx is a useful analytic tool for determining choles- terol in various samples: (a) total and esterified serum; (b) from low-density lipoproteins to high-density lipo- proteins; (c) on the cell membrane of erythrocytes (and of other cells and cellular compartments); and (d) in gall stones and in human bile. Normal human blood serum contains less than 5.2 mm (200 mgÆdL )1 ) of cholesterol; in plasma, lipoproteins contain cholesterol and about 70% is esterified by fatty acids. Determining the concentration of serum cholesterol is fundamental in the assessment of a variety of diseases (e.g. in atherosclerosis and other lipid disorders) and for estimating the risk of thrombosis, myocardial infarction, etc. The risk for Alzheimer disease is also related to hypercholesterol- emia via mechanisms involving oxidative stress: this disease is characterized by the accumulation of amyloid b-peptide (a 39–43 amino acid peptide) in the neocortex, which is connected to peroxidative damage. The amyloid b-peptide forms complexes with Cu 2+ ions, which oxidize cholesterol into cholest-4-en-3-one, thus mimicking the activity of ChOx. In fact, brain tissues from Alzheimer disease patients had a cholest- 4-en-3-one content  2-fold higher than brain tissues from controls [31]. The chemical methods for determining serum choles- terol (e.g. Liebermann–Burchard reactions) were ousted in 1973 by Richmond [32]: he had demonstrated that ChOx from Nocardia can be used to measure serum cholesterol by assaying the amount of hydrogen perox- ide produced. Hydrogen peroxide is reacted with quadri- valent titanium and xylenol orange, yielding a colored product measured at 550 nm. This method relied on the nonenzymatic hydrolysis (alkaline saponification) of the cholesterol esters. The same enzyme was then used for the serum cholesterol assay by directly measuring the absorbance of the product cholest-4-en-3-one at 240 nm, following a lengthy 2-hour incubation period and subsequent product extraction [33]. Total choles- terol could also be determined in serum (including the esters) by employing a totally enzymatic method in which cholesterol hydrolase (EC 3.1.1.13) was used to hydrolyze cholesterol esters [34]. Free cholesterol is then oxidized by ChOx to produce hydrogen peroxide, which is finally assessed enzymatically with horseradish peroxidase by the oxidative coupling of 4-aminoantipy- rine and phenol: this results in a quinoneimine dye (with an absorption maximum at 500 nm). This method is characterized by the advantages of simplicity and little interference, and is highly reproducible: it is still routinely used in clinical laboratories. The colorimetric method can also accurately determine serum cholesterol levels but, because of interference from pigments, it is difficult to assess bile cholesterol and thus an electro- chemical method based on oxygen consumption by ChOx reaction is better [35]. The different methods of determining serum cholesterol are reported in Table 2. Recently, the performance of the end point versus the kinetic method for enzymatic assay of cholesterol was compared [36]. The end point method is more accurate and precise; however, the kinetic method shows a lower sensitivity to interfering substances and analysis times are shorter. The method of Allain et al. [34] for assaying high- density lipoprotein cholesterol was improved by employing polyethylene glycol-modified ChOx and cholesterol esterase [37]. The serum, to which a-cyclo- dextran sulfate and a small amount of dextran sulfate was added, was directly analyzed without precipitating lipoprotein micelles, and the results compared favour- ably with the previous method. Subsequently, the same group reported on an automated method for measur- ing serum low-density lipoprotein cholesterol without ultracentrifugation separation [38]. This result was achieved by using a nonionic surfactant, polyoxyethyl- ene-polyoxypropylene block copolyether, magnesium ions and a sodium salt of sulfated cyclic maltohexaose, a-cyclodextrin sulfate. Polyethylene glycol-modified ChOx and cholesterol esterase were then used for the amperometric determination of high-density lipopro- tein cholesterol in 1–2 lL of serum [39]. For many years, ChOx was used in a great variety of biosensors based on different detection systems. For example, ChOx immobilized on a nylon membrane was used to build a fiberoptic biosensor based on the change in fluorescence of an oxygen-sensitive dye [40]; the analytical range is 0.2–3 mm, and the steady-state signal is achieved in 7–12 min. Subsequently, a choles- terol sensor based on the electrochemical reduction of oxygen was developed using a bilayer-film coating: this sensor is less sensitive to organic interferences [41]. ChOx was immobilized on nylon nets placed over the membrane of an oxygen probe or over the cellulose acetate membrane of a hydrogen peroxide detector and then covered with a polycarbonate membrane [42]. Biotechnological applications of sterol oxidase L. Pollegioni et al. 6862 FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS When incorporated in a flow injection system, the hydrogen peroxide-based device was fast, reproducible and linear over a 0.5–5 mm cholesterol concentration range. An amperometric biosensor was also obtained by reconstituting the apoprotein of Pseudomonas fluo- rescens ChOx with a FAD monolayer [43]: the sensor shows high sensitivity and selectivity towards electroac- tive interference. Most recently, self-assembled mono- layers have been used to improve response and binding in biosensors based on immobilized ChOx. The prop- erties of biosensors for determining cholesterol using ChOx immobilized by silane-based self-assembled monolayers onto an indium-tin oxide surface have been compared [44]. An amperometric-rotating biosen- sor using immobilized ChOx, cholesterol esterase and peroxidase was also produced; this biosensor contains a micropacked column with immobilized ascorbate oxidase to eliminate l-ascorbic acid interference [45]. By use of this device, cholesterol can also be deter- mined in the range of 1.2 lm–1 mm with a lifetime of 25 days of use. Furthermore, ChOx was also used to develop a sensor for determining total cholesterol in different food samples [46]. ChOx activity has also been employed to analyze cholesterol in cultured cells by means of an HPLC method. At first, the cholesteryl esters were quantita- tively hydrolyzed using cholesterol esterase, and then total cholesterol was converted by Nocardia ChOx to cholest-4-en-3-one before detection at 240 nm. In 3 min, free and total cholesterol can be measured in as few as 5000 cells of human monocyte-derived macrophages [47]. Subsequently, the same assay was developed using a fluorimetric method: cholesterol and related sterols have been assayed in sickle-cell and healthy erythrocytes [48]. Other examples of cholesterol analysis in cells have been briefly described [3]. Interestingly, the surface area of a cell can be easily estimated by using ChOx to deter- mine cell-surface cholesterol. For example, an area of 17500 lm 2 was calculated for human fibroblasts from the cholesterol concentration in the plasma membrane (equivalent to 44 fmolÆcell )1 ) and a known choles- terol ⁄ phospholipid molar ratio of 0.8 [49]. Table 2. Alternative ChOx-based methods for determining cholesterol concentration. Molecule assayed Method Detection limit Notes Reference Cholest-4-en-3-one HPLC with detection under UV light (240 nm) 0.15 nmol Determination of unsaponifiable lipids and thoracic duct lymph chylomicrons [75] Hydrogen peroxide Amperometric assay using immobilized ChOx and cholesterol esterase 1 lmolÆL )1 Determination of both free and total cholesterol in blood serum [76] Hydrogen peroxide Chemiluminescent detection of the reaction using lucigenin 2.6 lmolÆL )1 Requires accurate adjustment of the pH to 11.75–11.9 [77] Hydrogen peroxide Temperature-enhanced chemiluminescent flow system using bis-(2,4,6-trichlorophenyl) oxalate-perylene in the presence of Triton X-100 (detection at 500 nm) 19 lmolÆL )1 Significant increase in the signal-to-noise ratio [78] Hydrogen peroxide Fluorimetric determination (excitation 320 nm; emission 400 nm) using 4-hydroxyphenylacetic acid 0.5 nmolÆL )1 Measurements can be performed on 1 lL of serum [79] Hydrogen peroxide Spectrophotometric determination of the reaction with 4-aminophenazone plus phenol (at 520 nm) using immobilized ChOx, cholesterol esterase and horseradish peroxidase on arylamine glass beads 54 lmolÆL )1 The co-immobilized enzymes did not show loss of activity after 300 uses [80] Electron transfer from ChOx Electrochemical determination using electron mediators (i.e. 1-methoxy-5-methylphenazinium, thionine, etc.) 0.25–2.5 mmolÆL )1 The measurement is altered by the dissolved oxygen and requires ‡ 10 min to obtain a steady-state response. No effect of classical interference was observed in the colorimetric assays. [81] L. Pollegioni et al. Biotechnological applications of sterol oxidase FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS 6863 ChOx in agriculture Genetically modified plants that produce insecticidal proteins (e.g. the Bacillus thuringiensis toxin) are now available to control insect pests of several major crops. In 1993, from a random screening of > 10000 filtrates from microbial fermentations, Monsanto Co. (St Louis, MO, USA) discovered a highly efficient protein in cul- ture filtrates that killed boll weevil (Anthonomus grandis Boheman) larvae [50]. Histological and biochemical studies identified the protein as ChOx: purified ChOx is active against boll weevil larvae at a 50% lethal con- centration (LC 50 ) of 20.9 lgÆmL )1 , which is comparable to the bioactivity of B. thuringiensis proteins against other insect pests. The ChOx gene (Monsanto named it choM, see Fig. S1), from the Streptomyces sp. strain A19249, exhibits 85% DNA sequence identity and 89% amino acid sequence similarity with known Strep- tomyces sp. SA-COO (choA gene). In addition to boll weevil, several lepidopterans were negatively affected by the presence of ChOx at a dietary concentration of 0.001%. Adding the products of cholesterol oxidation by ChOx (i.e. cholest-en-3-one and hydrogen peroxide) to the diet, and pretreating the diet with the enzyme, excluded insecticidal effects caused by the ingestion of toxic compounds. However, the boll weevil larvae are acutely sensitive to ingested ChOx because it induces lysis at the midgut epithelium. Boll weevil adults are insensitive to ingested ChOx, although the fecundity of adult females was greatly reduced if 50 lgÆmL )1 of the enzyme was present in the diet [51]. ChOx reduced sub- sequent oviposition (up to 83% in eggs laid) and larval survival (97% reduction as compared to controls) because of poorly developed ovaries and few develop- ing oocytes. ChOx was expressed in transformed tobacco plants, and the synthesis levels in leaf tissues routinely ranged from approximately 5–50 lgof enzyme per g fresh weight. In the absence of a chloro- plast-targeting sequence, ChOx production resulted in severe abnormalities in plant development and fertility. When produced as a fusion with a chloroplast-targeting peptide, synthesis of the mature and the full-length enzyme did not cause the deleterious phenotypic effect observed with untargeted ChOx [52]. Transgenic leaf tissues expressing ChOx exerted insecticidal activity against boll weevil larvae. When produced in the cyto- sol, or when targeted to chloroplasts, ChOx metabo- lizes phytosterols in vivo. Transgenic plants expressing ChOx in cytosol accumulated low levels of saturated sterols (stanols), while the transgenic plants expressing chloroplast-targeted ChOx maintained a greater accu- mulation of stanols and appeared phenotypically and developmentally normal. It was proposed that ChOx could modify sterol ratios, thus influencing cell division, or could affect brassinosteroid biosynthesis in steroid hormones. ChOx in virulence ChOx is an interesting pharmaceutical target for treat- ing bacterial infections. R. equi is a Gram-positive coc- cobacillus that resides within macrophages of the host. It is a common soil organism that frequently infects young horses; the most common manifestation is a chronic suppurative bronchopneumonia with abscess formation and cavitary pneumonia (for a review, see [53]). Since 1967, this organism has also been reported to infect humans, being frequently diagnosed as an opportunistic pathogen in immunocompromised patients,  70% of whom are infected with HIV [54]. The clinical manifestations of R. equi infections are different: the most frequent form is severe pyrogranul- omatous pneumonia. Among the candidate virulence factors of this pathogenic actinomycete, in vitro data suggested that during R. equi infection of the host cell, membrane lysis is facilitated by the induction of extra- cellular ChOx [55]. Mutational analysis indicated that ChOx is the membrane-damaging factor responsible for the synergistic hemolytic reaction elicited by R. equi in the presence of sphingomyelinase C-produc- ing bacteria, such as Listeria ivanovii, Bacillus cereus and Staphylococcus aureus [55]. The membrane-damag- ing activity of R. equi requires the presence of bacterial sphingomyelinase C, thus indicating that the ChOx substrate is not directly accessible to the enzyme in intact membranes. A detailed description of the role of ChOx in the virulence of R. equi, as well as recently reported criticisms [56], has been reported previously [2]. ChOx as antifungal sensor Streptomyces natalensis ChOx (encoded by the pimE gene; Fig. S1) plays a main role in the biosynthesis of the polyene macrolide pimaricin [57] (the details of ChOx involvement in this biosynthetic process have been reported previously [2]). This 26-member tetraene macrolide antifungal antibiotic is widely used in the food industry (to prevent contamination of cheese and other nonsterile food with mold) and in the treatment of fungal keratitis because it interacts with membrane ster- ols (ergosterol is the major sterol found in fungal mem- branes), altering the membrane structure and causing cell leakage. Interestingly, putative ChOx-encoding genes are present in other known biosynthetic gene clus- ters of antifungal polyketides, such as filipin (pteG) and Biotechnological applications of sterol oxidase L. Pollegioni et al. 6864 FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS rimodicin ⁄ CE-108 (rimD). All these polyketides are elicited by soil bacteria against their fungal competitors, whose membranes contain ergosterol, representing a selective advantage for the producing organism. Protein engineering studies Improvement in thermal stability The temperature sensitivity of ChOxs from a number of sources has been compared recently [23]. The ther- mal stability (as well as the stability at alkaline pH) of ShChOx is not optimal for use as a diagnostic enzyme (discussed above). In order to improve its properties, a random mutagenesis approach by error-prone PCR of the choA gene was used [58]. Following screening of the mutant libraries at 50 °C, four single point mutants (S103T, V121A, R135H and V145E ShChOx, see the sequence numbering reported in Fig. S1) were isolated whose kinetic properties resemble those of the wild-type enzyme but whose thermal stability is increased. In par- ticular, the V145E ShChOx shows a marked increase in thermal stability and an enlarged range of optimal pH (from acid to alkali).The single mutations identified by random mutagenesis were combined. All the mutations, except for R135H, had an additive effect: the double mutant S103T ⁄ V145E was the most improved ShChOx because it shows unaltered activity after 4 weeks at 40 °C. Concerning the rationale of the observed changes, the C c atom of the threonine residue in S103T ShChOx seems to be accommodated in a relatively large cavity, thus strengthening the atomic packing (Fig. 2A). The V145E substitution introduces an addi- tional hydrogen bond and a salt bridge with the side chain of R417, a residue close to the FAD cofactor. These new interactions could help to stabilize the native conformation, thus increasing the thermal resistance and yielding a marked change in the optimal pH. BsChOx is a monomeric flavoenzyme containing one molecule of FAD covalently linked to H69 (mature protein numbering, corresponding to H121 on the full-length polypeptide; Fig. S1). The covalent link is eliminated by the H69A substitution: wild-type and H69A BsChOx do not show significant structural A B Fig. 2. (A) Details of the substitutions intro- duced in ShChOx (Protein Data Bank, PDB code: 1mxt) by random mutagenesis and resulting in a higher stability to temperature and to pH [58]. Mutated residues are col- ored according to the reporting authors as in Fig. S1. FAD (yellow) and 3-beta-hydroxy-5- androsten-17-one (AND, gray) are shown in CPK representation. Left: details of the regions surrounding two selected mutations, as reported by Nishiya et al. [58]. The molecular surface is shown in light brown. (B) Comparison of the active site of ReChOx (PDB code: 1coy) and the active site of ShChOx (PDB code: 1mxt), highlighting the position important for substrate specificity. Residues of the ReChOx active site are numbered as reported by Li et al. [68]; residues of the ShChOx active site are numbered as reported in Lario et al. [69] (representation licorice) to which were added residues mutated as described in Xiang & Sampson [64] (representation ball- and-stick). Proposed hydrogen bonds are represented in pink. The ligand AND was placed in the ShChOx active site according to the position occupied in the active site of ReChOx. L. Pollegioni et al. Biotechnological applications of sterol oxidase FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS 6865 differences [59]. The mutant enzyme retains the oxida- tion and the isomerization catalytic activities. How- ever, the H69A BsChOx shows a 35-fold decrease in maximal oxidation activity and a flavin midpoint reduction potential of  100 mV lower ()204 mV ver- sus )101 mV for wild-type ChOx) [60]. We demon- strated that the covalent bond of the flavin in ChOx represents a structural device for stabilizing the protein tertiary structure [61]. In fact, the urea-induced unfold- ing of H69A BsChOx occurred at significantly lower urea concentrations ( 2 m lower), as did the tempera- ture-induced unfolding ( 10–15 °C lower), than for the wild-type enzyme. Alteration of the substrate specificity Table 1 describes the substrate preference of some ChOxs (expressed as relative activity with respect to cholesterol). In vivo random mutagenesis of the choA gene decreased the K m value for cholesterol by 10-fold without changing maximal activity: the evolved ShChOx mutant contained the V145E ⁄ G405S substitu- tions [62]. Based on sequence comparison between Streptomyces and Rhodococcus ChOxs, six positions were mutated in ShChOx [63]. Among the mutants generated, the S379T variant showed a twofold higher activity towards pregnenolone than cholesterol as a substrate (interestingly, a threonine is present in the same position in ReChOx, i.e., T387; see Fig. 2B). The catalytic efficiency (k cat ⁄ K m ) of this mutant enzyme for cholesterol and pregnenolone was two- and sixfold higher, respectively, than that of the wild-type enzyme [63]. Noteworthy is that the cavity around the side chain of S379 in ShChOx is larger than that surround- ing T387 in ReChOx (Fig. 2B). Structure-based rational design of ShChOx was also used to identify the residues involved in substrate binding [64]: M58, L82, V85, M365, and F433 were identified as being in direct contact with the C17 sterol tail (these residues correspond to M95, L119, V122, M402 and F470 in the full-length sequence reported in Fig. S1). Each position was degenerated using NYS (N = A, C, G, T; Y = C, T; S = C, G) codons to limit amino acids to predominantly hydrophobic residues. The L82A ⁄ V85T ⁄ F433L ShChOx mutant showed a twofold increase in the k cat ⁄ K m ratio with b-sitosterol and stig- masterol, which differ from cholesterol for the extended C17 tail. By deleting the five residues belonging to the active-site loop in ShChOx (mutant D79-83 corre- sponding to sequence 115-119 in Fig. S1), the kinetic efficiency (k cat ⁄ K m ratio) was increased 5.6-fold for dehydroepiandrosterone compared with the wild-type enzyme, while the same ratio decreased 170-fold with micellar cholesterol and up to 2800-fold with choles- terol in a vesicle [2]. These results also suggest that the tip of the active-site loop is necessary for the packing with the C17-tail, being mainly responsible for the substrate specificity at this position of ChOx [65]. The oxidation and isomerization reactions catalyzed by ShChOx could be separated by site-directed muta- genesis. The N480A ⁄ Q mutants (corresponding to position N522 in the full-length sequence reported in Fig. S1) showed no oxidative activity but retained the ability to isomerize cholest-5-en-3-one into cholest-4- en-3-one [66]. However, the E361D ShChOx mutant (corresponding to the E356D mutant in [66] and to position 398 in the full-length sequence; Fig. S1) retained the oxidative activity, but not the isomeriza- tion activity [67]. Furthermore, the k cat ⁄ K m ratio for the oxidation reaction was increased sixfold in the E356D ShChOx mutant and the k cat ⁄ K m ratio for the isomerization reaction was increased threefold in the N480A mutant. Conclusions Certainly, future molecular biology, biochemical and structural investigations will enable us to clarify the role that ChOx activity plays in different microorgan- isms and also to identify new functional roles for this flavooxidase. Furthermore, in order to comprehend why this enzyme exists as a flavoprotein containing noncovalently or covalently linked FAD, we will also need to understand its physiological role. These inves- tigations will be carried out whilst optimizing known applications of the ChOx reaction and developing new, and more sophisticated, biotechnological uses of this flavoenzyme. The latter hinges both on the discovery of novel ChOx activities (see the recent identification of ChOx from thermophilic bacteria) and on the engi- neering of enzyme variants. We assume that the num- ber of investigations of ChOx will continue to grow quickly in the near future, in particular to identify unexpected areas of application. Acknowledgements This work was supported by grants from Fondo di Ateneo per la Ricerca. We are grateful for the support from Consorzio Interuniversitario per le Biotecnologie (CIB) and from the Research Center of Biotecnologie per la Salute Umana. We would like to apologize to many colleagues whose work we could not discuss in detail because of space limitations. Biotechnological applications of sterol oxidase L. Pollegioni et al. 6866 FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... 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MINIREVIEW Cholesterol oxidase: biotechnological applications Loredano Pollegioni, Luciano Piubelli and Gianluca Molla Dipartimento di Biotecnologie. assay of total and free cholesterols in serum by the combined use of immobilized cholesterol esterase and cholesterol oxidase reactors and peroxidase electrode