Biochemical and Biophysical Research Communications 399 (2010) 347–351
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Giardia lamblia encodes a functional flavohemoglobin Steven Rafferty a,*, Betty Luu b, Raymond E. March a, Janet Yee b,* a b
Department of Chemistry, Trent University, 1600 West Bank Drive, Peterborough, ON, Canada K9J 7B8 Department of Biology, Trent University, 1600 West Bank Drive, Peterborough, ON, Canada K9J 7B8
a r t i c l e
i n f o
Article history: Received 14 July 2010 Available online 22 July 2010 Keywords: Giardia Flavohemoglobin Nitric oxide Heme
a b s t r a c t Giardia lamblia is a pathogenic protist that infects the small intestine of mammals. As a facultative anaerobe, Giardia obtains all of its energy by substrate-level phosphorylation, lacks a functioning respiratory chain, and is not thought to require heme. However, sequencing of the G. lamblia genome has identified several putative heme proteins, one of which shares high sequence similarity to flavohemoglobins found in bacteria and some single-celled eukaryotes. We have cloned and characterized the functional properties of the G. lamblia flavohemoglobin. The protein is monomeric, binds heme and flavin adenine dinucleotide, and exhibits similar behavior to known flavohemoglobins, including NADH and NADPH oxidase activity, which is stimulated by addition of the nitric oxide donor DEA/NO. Based on its structural and functional properties, the likely role of this protein is to protect Giardia against oxygen, nitric oxide, or both. The presence of a Giardia gene encoding a functional heme protein raises questions on how this organism acquires the heme cofactor, which hitherto have been unexplored. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Giardia lamblia is a protozoan parasite that is endemic to freshwaters worldwide, and has been designated as a Category B Priority Pathogen by the Centres for Disease Control and the National Institutes of Allergy and Infectious Diseases. Ingestion of infectious Giardia cysts by humans, livestock or wildlife leads to their differentiation into free-swimming trophozoites, which multiply and adhere to the epithelial lining of the small intestine. Giardia infection is associated with severe abdominal cramps, diarrhea, and dehydration, among other symptoms. Passage of Giardia trophozoites into the large intestine triggers encystation, and the resulting cysts are excreted in the feces. Giardia is an aerotolerant anaerobe, and its energy is obtained through anaerobic glycolysis and other forms of substrate-level phosphorylation [1]. As a parasite, Giardia relies on its host, and specifically on the contents of the host’s digestive tract, for many of its cellular functions. In spite of its anaerobic metabolism, Giardia consumes molecular oxygen through the use of one or more flavoproteins, which use electrons provided by NADH to reduce oxygen to water [2]. Although Giardia have mitosomes, which are highly reduced mitochondria-like organelles used for iron–sulfur cluster assembly, they lack the respiratory chain found in the mitochondria of aerobic eukaryotes [3]. Consequently, oxygen
Abbreviations: DEA/NO, diethylamine NONOate; gFLHb, Giardia flavohemoglobin. * Corresponding authors. Fax: +1 705 748 1625. E-mail addresses:
[email protected] (S. Rafferty),
[email protected] (J. Yee). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.07.073
reduction in Giardia is not coupled to ATP production but rather protects this organism from oxidative stress, and regenerates oxidized nicotinamide cofactors required for substrate-level phosphorylation. The Giardia genome is compact (11.7 Mb) and has been the subject of a large scale sequencing project [4,5]. Among the surprises revealed by this work is the presence of a gene homologous to those that encode flavohemoglobins, and it is likely that Giardia acquired it via a lateral gene transfer from a bacterium, most likely a Vibrio sp. [5,6]. Its presence in Giardia is unusual as this organism lacks the enzymes required for heme biosynthesis and, indeed, lacks heme proteins such as cytochromes, peroxidases, and cytochrome P450-type enzymes. Flavohemoglobins are found in many bacteria and several unicellular eukaryotes; several structures from this enzyme class have been determined [7]. These proteins have an amino-terminal heme-binding globin domain linked to a carboxy-terminal FAD and NAD(P)H binding flavin domain. Although these proteins are able to reduce heme-bound oxygen to water, they are more efficient at catalyzing the oxidation of nitric oxide (NO) to nitrate by ferrous oxygenated heme, which leaves heme in the ferric state. The catalytic cycle is completed by the reduction of the ferric heme with electrons supplied from NAD(P)H via FAD, followed by oxygen binding [8]. As nitric oxide at high micromolar levels is toxic to Giardia [9], a functional flavohemoglobin may help protect it from the damaging effects of this free radical. To determine whether the sequence found in the Giardia genome encodes a functional protein, we cloned this gene into a bacterial expression vector. The protein
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was expressed at high levels with both heme and FAD cofactors bound, and has NADH oxidase activity, which is enhanced in the presence of NO.
2. Materials and methods The genomic DNA sequence of gFLHb (gene ID GL50803_15009) was obtained from the Giardia database (http://GiardiaDB.org). The coding sequence was PCR-amplified from Giardia genomic DNA using the following primer set: Forward primer (NdeI site underlined, extra nucleotides in lower case), 50 tcgaaagg CATATG ACG CTT TCC GAA GAC ACC CTG AG 30 ; Reverse primer (BlpI site underlined, extra nucleotides in lower case), 50 ttatactc GCTCAGC CTA ATG GGA GGC CTT GAA GGG GCC 30 . The amplicon was digested with NdeI and BlpI at 37 °C, and ligated to the pET14b plasmid vector that was previously digested with the same restriction enzymes and dephosphorylated with shrimp alkaline phosphatase. All enzymes were purchased from New England Biolabs. The resulting vector, pET14b-H6-gFLHb, was used to transform electrocompetent BL21(DE3) Escherichia coli cells. DNA sequencing was performed on the plasmid DNA isolated from one of these bacterial colonies to ensure the correct sequence and insertion of the Giardia flavohemoglobin gene (gFLHb). A single bacterial colony was used to inoculate a 5 mL culture of LB + ampicillin (50 lg/ mL), which was then incubated at 37 °C overnight. Four 2 L culture flasks, each containing 400 mL of LB + ampicillin were each inoculated with 1 mL of the small-scale culture, and incubated at 37 °C with shaking for one to two days. Cells were pelleted at 4000g for 10 min at 4 °C, and resuspended in 50 mL binding buffer (0.05 M Tris–HCl, 0.5 M NaCl, pH 7.5) supplemented with protease inhibitor cocktail (Bioshop Canada, Burlington, ON). The cell suspension was placed on ice, and subjected to sonication. The cell lysate was centrifuged at 12,000g for 30 min yielding a red-orange colored supernatant, which was then applied to a 5 mL His-Trap HP column (GE Health Sciences, Baie d’Urfe, QC). The column was washed with 25 mL binding buffer, followed with 25 mL wash buffer (0.05 M Tris–HCl, 0.5 M NaCl, 25 mM imidazole, pH 7.5). Finally, the recombinant protein was collected by the addition of elution buffer (0.05 M Tris–HCl, 0.5 M NaCl, 250 mM imidazole, pH 7.5). The protein was concentrated and exchanged into binding buffer on a 15 mL Amicon ultrafiltration unit with a 30 kDa MW cut off. An aliquot of the protein was analyzed by SDS–PAGE. Heme content was determined by the pyridine hemochrome assay [10], and protein concentration by DC assay. Flavin was quantified by boiling a portion of protein in 0.05 M potassium phosphate buffer, pH 7.5 for three minutes, followed by centrifugation of the sample (12,000 RPM on an Eppendorf microcentrifuge) and comparing its spectrum to that of a reference solution of FAD. To determine the quaternary structure of the protein, size exclusion chromatography on a Superdex 200 column was performed in 0.05 M Tris–HCl, 0.15 M NaCl, pH 7.5 at a flow rate of 0.5 mL/min. Electrospray ionization/mass spectrometric (ESI-MS) analysis was performed on a Quattro triple-stage quadrupole mass spectrometer (Micromass, Manchester, UK). The sample was admitted by direct infusion at 10 lL min1 using a model 11 syringe pump (Harvard Apparatus, Holliston, MA, USA). The mass range of m/z 600–1200 was recorded with unit mass resolution. All data acquisition and processing were carried out using Masslynx NT 3.5, from Micromass. Spectroscopic and enzyme assay measurements were determined on a Cary 400 Bio UV–visible spectrophotometer. The reduced oxygenated species was formed by addition of 100 lM NADH to the protein under aerobic conditions while the reduced deoxygenated species was generated by the addition of excess sodium dithionite to the protein in a stoppered cuvette. For rate measurements, NADH oxidation was monitored by the decrease in
absorbance at 340 nm over time. Baseline levels of NADH oxidation were measured prior to the addition of flavohemoglobin to the assay mixture. Assay conditions were 25 °C, 0.05 M potassium phosphate, pH 7.5, 100 lM NAD(P)H, and 1 lM FAD when present. For measurements at pH 6.5, 0.05 M Bis–Tris buffer was used. The amount of flavohemoglobin in the assay was quantified based on heme content. For NADH oxidation measurements in the presence of an NO donor, initial rates were measured in the presence of NADH alone, followed by addition of 100 lM DEA/NO (Cayman Chemical, Ann Arbor, MI). Flavin fluorescence measurements were determined with an excitation at 460 nm and the emission spectrum between 480 and 600 nm was recorded on a Shimadzu RF5000 spectrofluorimeter. Secondary structure predictions of the pair of unique sequence inserts in the Giardia flavohemoglobin were determined with the program GOR4 [11]. 3. Results and discussion Bacterial cultures expressing recombinant gFLHb yielded redbrown cell pellets upon centrifugation and dark-red supernatants upon cell lysis; the yield of purified gFLHb was 100–120 mg per liter of E. coli culture. Based on the pyridine hemochrome assay for heme and fluorimetric assays for FAD, the protein was isolated with 0.65 equivalents of heme and 0.3 equivalents of FAD extracted per protein chain. Although these values are substoichiometric, and were obtained without including the heme precursor aminolevulinic acid or the FAD precursor riboflavin in the culture media to maximize cofactor content [12], these cofactor levels are well within the range observed with analysis of recombinant flavohemoglobins from other organisms [12]. The protein is monomeric with a molecular weight of 54 kDa.
Fig. 1. UV–visible spectra of gFLHb. Black trace, ferric enzyme, as isolated. Red trace, NADH-reduced under aerobic conditions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 1 UV–visible spectroscopic features of gFLHb. All spectra were measured in 0.05 M potassium phosphate buffer, pH 7.5, 25 °C. Shoulders are indicated by ‘‘s”. Complex
kmax (nm)
Fe(III) Fe(III)–NO Fe(III)–imidazole Fe(II)–O2 Fe(II) deoxy
403–405, 450–490(s), 533(s), 645 417, 486(s), 534, 566 413–414, 540, 570(s) 416, 545, 580 433
S. Rafferty et al. / Biochemical and Biophysical Research Communications 399 (2010) 347–351 Table 2 NADH oxidation rate data for gFLHb. Conditions
Rate of nicotinamide cofactor oxidation (min1)
NADH, pH 7.5 NADH, FAD, pH 7.5 NADPH, FAD, pH 7.5
2.6 ± 0.1 6.5 ± 0.1 2.2 ± 0.1
NADH, DEA-nonoate, pH 7.5 NADH, FAD, DEA-nonoate, pH 7.5
5.6 ± 0.1 9.8 ± 0.1
NADH, FAD, pH 6.5 NADH, FAD, DEA-nonoate, pH 6.5
13.6 ± 0.1 34.4 ± 0.1
Conditions: 100 lM NAD(P)H, 1 lM FAD (when present), 100 lM DEA-nonoate (when present), 25 °C.
100 µM DEA/NO
0.6
0.4
0.2
0 0
1
2
3
Time, minutes Fig. 2. Rate of NADH oxidation by gFLHb. The reaction was initiated by adding enzyme at time zero to a solution of 100 lM NADH, 1 lM FAD, 0.05 M Bis–Tris, pH 6.5, 25 °C. After establishing the initial rate of oxidation, 100 lM DEA/NO was added (arrow).
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The gFLHb protein is purified in its ferric state and appears to be a mixture of high- and low-spin species based on the asymmetric Soret band centered at 403 nm (Fig. 1). The association of flavin with this protein is indicated by the presence of the low shoulder between 460–480 nm in the spectrum (Fig. 1), as well as a strong fluorescence emission at 520 nm when excited at 460 nm (data not shown). Addition of NADH generates a characteristic Fe(II) oxyheme spectrum, and bleaches the shoulder at 460–480 nm, which is consistent with electron transfer mediated through the FAD cofactor (Fig. 1). NADH also reduces flavin as indicated by a loss of FAD-associated fluorescence, which is eventually restored as NADH is oxidized (data not shown). Key spectroscopic features of the protein in different oxidation and ligation states are summarized in Table 1; these properties of the Giardia flavohemoglobin are typical of other members of this enzyme class [13,14]. Giardia flavohemoglobin possesses NADH oxidase activity, which is enhanced by the presence of FAD in the assay solution, and is pH-dependent (Table 2). The enzyme can also use NADPH as a reducing agent, although with lower activity. There is a pronounced increase in the rate of NADH oxidation when the NO-donor compound DEA/NO is added to the assay (Fig. 2), which is consistent with either nitric oxide dioxygenase activity or nitric oxide reductase activity. However, it is more likely that the increased NADH oxidation rate is due to nitric oxide dioxygenase activity because this is the innate function of flavohemoglobins, and the assays were performed under aerobic conditions. The rate of NADH oxidation under these conditions seems to be limited only by the rate of release of NO from the donor complex, which, at pH 7.4, has a half-life of about 16 min that decreases markedly with increasing pH [15]. The enhancement of NADH oxidation was not due to the breakdown products of DEA/NO, as solutions that were assayed for activity one hour after the addition of this NO donor showed nearly basal levels of NADH oxidation (data not shown).
Fig. 3. Sequence alignment of gFLHb with two flavohemoglobins for which tertiary structures have been determined by X-ray diffraction. AeFLHb: Alcaligenes eutrophus flavohemoglobin; Hmp: E. coli flavohemoglobin. The insertions in the globin and the FAD domains in gFLHb are underlined and indicated by blue and red text, respectively. Asterisks indicate identical residues, while colons and periods indicate conserved residues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. The cartoon structure of E. coli flavohemoglobin Hmp (1GVH.PDB [16]). The location of the 21-residue inserts in the globin domain (blue) and 29-residue insert in the flavin domain (red) that occurs in gFLHb are mapped onto the E. coli structure and are indicated by arrows. Heme is shown in cyan and FAD in orange; helices E and H of the globin domain are also noted. MacPyMol (Delano Scientific) was used to generate this representation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
The structure of the Giardia flavohemoglobin retains many of the highly conserved residues found in the active site of other family members, but it also has unique sequence elements such as a 21 amino acid insertion in the N-terminal globin domain and a 29 amino acid insertion in the C-terminal FAD-binding domain (see underlined residues in Fig. 3). BLAST searches for these inserted sequences did not find them in any other flavohemoglobin family members. When mapped onto the structure of the E. coli flavohemoglobin, both of these inserts are located on the same face of the enzyme (Fig. 4), but their structures and functional significances are unknown. The insertion in the globin domain of gFLHb (indicated in blue in Fig. 4) occurs at the turn between a-helices E and F, and does not have any strong a-helix or b-strand characteristics. The insertion in the FAD domain (indicated in red in Fig. 4) occurs in a loop that links the FAD and NADH binding modules and partially contacts helix F within the globin domain. Secondary structure analysis of this insert suggests that it can form a 14-residue a-helix followed by a 5-residue b-strand. These additional structural elements may affect the relative orientation of the globin and flavin domains within gFLHb. By comparison, E. coli and Alcaligenes eutropha flavohemoglobins contain insertions and deletions
