Role of the Phenylalanine Hydroxylating System in Aromatic Substance Degradation and Lipid ...

46 Pages·2013·1.56 MB·English

AEM Accepts, published online ahead of print on 15 March 2013
Appl. Environ. Microbiol. doi:10.1128/AEM.00238-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
1 Role of the Phenylalanine Hydroxylating System in Aromatic Substance
2 Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella
3 alpina
4 Running title: Phenylalanine Hydroxylating System in M. alpina
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6 Hongchao Wanga,b, Haiqin Chenb＃, Guangfei Haoa,b, Bo Yanga,b, Yun Fengc, Yu Wanga,b, loa
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7 Lu Fengc, Jianxin Zhaob, Yuanda Songb, Hao Zhangb, Yong Q. Chena,b, Lei Wangc, Wei d

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8 Chena,b＃

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10 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi m
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11 214122, P.R. Chinaa m
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12 School of Food Science and Technology, Jiangnan University, Wuxi 214122, P.R. Chinab g
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13 TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin A
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14 Economic-Technological Development Area, Tianjin 300457, P. R. Chinac il 4
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16 ＃Corresponding authors: Haiqin Chen and Wei Chen y

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17 Mailing address: 1800 Lihu Ave, Wuxi, Jiangsu 214122, P.R. China. es
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18 Phone: [phone]. Fax: [phone].
19 E-mail: [email protected]; [email protected]
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21 Journal section: Genetics and molecular biology
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22 Abstract
23 Mortierella alpina is a filamentous fungus commonly found in soil, which is able to
24 produce lipids in the form of triacylglycerols that account for up to 50% of its dry weight.
25 Analysis of the M. alpina genome suggests that there is a phenylalanine hydroxylating
26 system for the catabolism of phenylalanine, which has never been found in fungi before. D
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27 We characterized the phenylalanine hydroxylating system in M. alpina to explore its role n
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28 in phenylalanine metabolism and its relationship to lipid biosynthesis. Significant de
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29 changes were found in the profile of fatty acids in M. alpina grown on medium ro
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30 containing an inhibitor of the phenylalanine hydroxylating system compared to M. alpina t
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31 grown on medium without inhibitor. Genes encoding enzymes involved in phenylalanine a
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32 hydroxylating system (phenylalanine hydroxylase, pterin-4α-carbinolamine dehydratase, a
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33 and dihydropteridine reductase) were expressed heterologously in Escherichia coli and o
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34 the resulting proteins were purified to homogeneity. Their enzymatic activity was o
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35 investigated by HPLC or VIS-UV spectroscopy. Two functional phenylalanine r
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36 hydroxylase (PAH) enzymes were observed, encoded by distinct gene copies. A novel 2
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37 role for tetrahydrobiopterin in fungi as a cofactor for PAH is suggested, which is similar
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38 to its function in higher life forms. This study establishes a novel fungal scheme for the u
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39 degradation of an aromatic substance (phenylalanine) and suggests that the phenylalanine
40 hydroxylating system is functionally significant in lipid metabolism.
41 Key words: M. alpina, phenylalanine hydroxylating system, phenylalanine degradation,
42 amino acid metabolism, lipid metabolism
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43 INTRODUCTION
44 The phenylalanine hydroxylating system catalyzes the irreversible hydroxylation
45 of phenylalanine to tyrosine, which is the rate-limiting step in animal phenylalanine
46 catabolism and protein and neurotransmitter biosynthesis, and results in the formation
47 of one molecule of fumarate and one of acetyl-CoA from each molecule of D
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48 phenylalanine (1, 2). The animal phenylalanine hydroxylating system consists of n
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49 several essential components: phenylalanine hydroxylase (PAH; EC1.14.16.1), d
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50 pterin-4α-carbinolamine dehydratase (PCD; EC4.2.1.96), dihydropteridine reductase ro
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51 (DHPR; EC1.5.1.34), and the obligatory cofactors tetrahydrobiopterin (BH4) and ht
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52 molecular oxygen (3, 4) (Fig. 1). PAH is one of the BH4-dependent aromatic amino //a
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53 acid hydroxylases, which also include tryptophan hydroxylase (TrpOHase; .
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54 EC1.14.16.4) and tyrosine hydroxylase (TyrOHase; EC1.14.16.2) (5). BH4 is .
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55 regenerated following PAH-mediated phenylalanine hydroxylation by two additional /

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56 enzymes, PCD and DHPR (Fig. 1) (6). The PAH, PCD, and DHPR genes responsible A
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57 for the phenylalanine hydroxylating system in animals have already been il 4
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58 characterized (7, 8). 0
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59 Phenylalanine is an important aromatic compound, one of the structurally diverse y

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60 and second most abundant class of organic substrates (9). The greatest challenge for e
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61 organisms using aromatic compounds as growth substrates is the stabilizing resonance
62 energy of the aromatic ring system (10). This aromatic structure makes the substrate
63 unreactive to oxidation or reduction, and thus requires elaborate degradation strategies
64 (9). The degradation of aromatic substances is dominated by aerobic and anaerobic
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65 bacteria and aerobic fungi, and the strategies used by these organisms are quite
66 different from those of animals (10). Unlike higher organisms, most bacteria, fungi,
67 and plants do not convert phenylalanine into tyrosine. The presence of PAH has only
68 been reported in a few bacteria based on the identification of related genes (11-14). In
69 fungi Aspergillus nidulans and Aspergillus fumigatus, tyrosine was thought to be be D
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70 synthesized from phenylalanine by PAH (15, 16); however, no gene encoding PAH n
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71 was found in their genomes (Table S1). To date, no PAH, PCD, or DHPR involved in d
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72 the phenylalanine hydroxylating system has been identified in any fungi. ro
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73 Phenylalanine is converted into phenylacetate in Penicillium chrysogenum, h
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74 Aspergillus nidulans, and Aspergillus niger (Fig. 2A), or into cinnamic acid in //
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75 Bjerkandera adusta and Schizophyllum commune (Fig. 2C) (17-20). The cinnamate .
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76 thus formed is converted to protocatechuate through benzoate, whereas
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77 phenylpyruvate is converted to homogentisate, which is catabolized by cleavage of /

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78 the aromatic ring to yield fumarate and acetyl-CoA. Recently, we sequenced the A
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79 whole genome of M. alpina (ATCC 32222) (21), and our analysis suggested that there il 4
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80 are two putative copies of BH4-dependent PAH gene for the catabolism of 01
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81 phenylalanine in M. alpina. Genes encoding PCD and DHPR, which are required for y

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82 the regeneration of BH4, an essential component of the phenylalanine hydroxylating e
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83 system (6), were also found in the M. alpina genome. The BH4 de novo synthesis
84 pathway in M. alpina was first purified and characterized in our laboratory (22), but
85 the function of BH4 in fungi is still not well understood. The presence of the
86 phenylalanine hydroxylating system in M. alpina suggests that fungi can make use of
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87 this system in their phenylalanine degradation strategy.
88 Mortierella alpina is a well-known polyunsaturated fatty acid (PUFA) producing
89 oleaginous fungus commonly found in soil (23). Some of the genes necessary for lipid
90 synthesis in M. alpina have been cloned and partially characterized (24-28), and
91 several biochemical reactions have been studied in detail (29, 30). However, the D
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92 molecular mechanism of efficient lipid biosynthesis is still not well understood in n
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93 oleaginous fungi in general, and in M. alpina in particular. PAH and its cofactor BH4 de
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94 have been suggested to be essential for lipid metabolism in higher organisms (31-36), ro
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95 and some amino acid metabolism pathways have been postulated to be involved in h
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96 fatty acid biosynthesis in oleaginous fungi (37, 38). However, the functional //
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97 significance of the phenylalanine hydroxylating system in the biosynthesis of lipids .
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98 and closely related compounds has yet to be fully elucidated. In higher organisms, the
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99 phenylalanine hydroxylating system is inhibited by p-chlorophenylalanine or esculin. /

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100 Both are complete and irreversible inhibitors of PAHs in vivo (39, 40). M. alpina is A
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101 noteworthy for its production of PUFA de novo, and due to its high lipid content (23) il 4
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102 provides an interesting model for studying the relationship between the phenylalanine 0
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103 hydroxylating system and lipid metabolism. y

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104 In this study, we investigate the role of the phenylalanine hydroxylating system e
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105 in lipid metabolism, and probed its possible function in the degradation of aromatic
106 substances. We establish a novel role for BH4 in fungi, similar to that known to exist
107 in higher life forms. We characterized the genes encoding PAH, PCD, and DHPR and
108 their functions in the phenylalanine hydroxylating system in vitro. We identified two
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109 functional PAH genes (PAH-1 and PAH-2) and measured kinetic parameters and the
110 effects of temperature, pH and aromatic amino acids on PAHs activity. Multiple
111 sequence alignment and phylogenetic analysis of the PAH proteins with other
112 homologous proteins were also performed.
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116 MATERIALS AND METHODS ro
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117 Gene search. Predicted proteins in the M. alpina (ATCC 32222) genome h
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118 (GenBank accession number ADAG00000000) were annotated by BLAST (41) //
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119 searches against the following protein databases with the E-value 1E-5: NR .
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120 (www.ncbi.nlm.nih.gov), KOGs and COGs (42), KEGG(43), Swiss-Prot, and
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121 UniRef100 (44), BRENDA (45), and by InterProScan (46) using the default parameter /

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122 settings. Pathway mapping was conducted by associating the EC assignment and KO A
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123 assignment with the KEGG metabolic pathways based on the BLAST search results. il 4
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124 The predicted PAH-1, PAH-2, PCD, and DHPR proteins in the M. alpina genome 0
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125 were searched against predicted proteins of sequenced fungal genomes by BLAST y

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126 with the E-value 1E-5. e
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127 Strains and growth conditions. M. alpina (ATCC 32222) was cultured on
128 potato dextrose agar at 25ºC for 5–7 days. The fungal cultures were initially cultivated
129 in 200 ml of Kendrick medium (47) and incubated at 25ºC for 6 days. The mycelia
130 were then collected by filtration through sterile cheesecloth, and frozen immediately
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131 in liquid nitrogen for RNA extraction.
132 Aromatic compound degradation test. The indicated sole carbon sources
133 (glucose at 3%, phenylalanine at 25 mM, phenylacetate at 10 mM and Tyrosine at
134 25 mM) (15) were added to minimal medium to test the aromatic compound
135 degradation in M. alpina. The minimal medium contained 0.3% diammonium tartrate, D
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136 0.7% KH2P04, 0.2% Na2HP04, 0.15% MgS04·7H20, 0.67% yeast nitrogen base and nlo
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137 2% agarose, pH 6.0. The plates were photographed after 3 days of incubation at 25°C. d
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138 The mycelia of M. alpina were stained with 0.01% triphenyltetrazolium chloride ro
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139 (TTC) to facilitate growth measurements (48). This experiment was replicated three h
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140 times. //
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141 Effect of a PAH inhibitor on lipids synthesis. For the whole-cell inhibition .
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142 studies, cultures were grown in 50 ml of Kendrick medium (50 g/l glucose, 2 g/l
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143 diammonium tartrate, 7.0 g/l KH2PO4, 2.0 g/l Na2HPO4, 1.5 g/l MgSO4.7H20, 1.5 g/l /
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144 Bacto yeast extract, 0.1 g/l CaCl22H2O, 8 mg/l FeCl3.6H2O, 1 mg/l ZnSO4.7H2O, 0.1 A
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145 mg/l CuSO4.5H2O, 0.1 mg/l Co(NO3)2.6H2O and 0.1 mg/l MnSO4.5H2O, pH 6.0) with il 4
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146 the addition of 5 mM p-chlorophenylalanine. The medium and incubation protocols 0
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147 were as previously described (21). The lipid composition of M. alpina was also y

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148 investigated when p-chlorophenylalanine (5 mM) and tyrosine (5 mM) were both e
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149 added to the medium. M. alpina cultures were incubated at 25°C for 6 days.
150 Aspergillus oryzae is another oleaginous fungus (49, 50), and genome analysis
151 suggests there is no phenylalanine hydroxylating system in A. oryzae (Table S2). The
152 addition of PAH inhibitor in A. oryzae was included to verify whether there were any
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153 changes in lipids in the absence of a phenylalanine hydroxylating system. A. oryzae
154 was incubated at 25°C for 6 days. The mycelia were then collected by filtration and
155 approximately 20 mg was used for each lipid extraction. Accurately weighed portions
156 of pulverized mycelium were extracted using the method of Bligh and Dyer (51)
157 under acidified conditions with pentadecanoic acid and heneicosanoic acid added as D
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158 internal standards (21). This experiment was replicated 3 times. n
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159 Cloning and plasmid construction. Total RNA extraction was performed using de
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160 Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. RNA was ro
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161 subjected to RNase-free DNase digestion and then purified using the RNeasy Mini kit h
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162 (Qiagen). The quantity and quality of the total RNA were evaluated using a NanoDrop //
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163 ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). The total RNA was .
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164 reverse transcribed with the PrimeScript RT reagent kit (Takara Bio, Inc.) following
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165 the manufacturer’s instructions, before the PCR amplification of PAH-1, PAH-2, PCD, /

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166 and DHPR genes using the primer pairs shown in Table 1. The PCR conditions used A
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167 were as follows: denaturation at 95ºC for 30 s, annealing at 50ºC for 45 s, and il 4
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168 extension at 72ºC for 1 min (for 25 cycles in total). The final volume in each well was 0
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169 50 µl. The amplified product was cloned into pET28a+ to construct pwl47864 by

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170 (containing PAH-1), pwl47866 (containing PAH-2), pwl39696 (containing PCD), or e
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171 pwl39698 (containing DHPR). The presence of inserts in the plasmids was confirmed
172 by sequencing using an ABI 3730 Sequencer.
173 Protein expression and purification. E. coli BL21 carrying pwl47864,
174 pwl47866, pwl39696, or pwl39698 was grown overnight at 37ºC with shaking in LB
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175 medium containing 50 μg/ml of kanamycin. The overnight culture (10 ml) was
176 inoculated into 500 ml of fresh medium and grown until the OD600 reached 0.6.
177 PAH-1 expression was induced by the addition of 0.1 mM IPTG at 30ºC for 20 h,
178 PAH-2 expression was induced by 0.01 mM of IPTG at 30ºC for 20 h, and PCD or
179 DHPR expression was induced by 0.5 mM of IPTG at 37ºC for 16 h. After the IPTG D
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180 induction, cells were harvested by centrifugation, washed with binding buffer (50 mM n
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181 Tris-HCl, 300 mM NaCl, and 10 mM imidazole; pH 8.0), resuspended in the same d
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182 buffer (supplemented with 1 mM phenylmethanesulfonyl fluoride and 1 mg/ml ro
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183 lysozyme), and then sonicated. Cell debris were removed by centrifugation, and the h
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184 supernatants (containing the soluble proteins) were collected. The His-tagged fusion //
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185 proteins were purified by nickel ion affinity chromatography using a Chelating .
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186 Sepharose Fast Flow column (GE Healthcare) according to the manufacturer’s
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187 instructions. Unbound proteins were washed through with 100 ml of wash buffer (50 /

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188 mM Tris-HCl, 300 mM NaCl, and 25 mM imidazole; pH 8.0). Fusion proteins were A
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189 eluted with 3 ml of elution buffer (50 mM Tris-HCl, 300 mM NaCl and 250 mM il 4
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190 imidazole; pH 8.0) and dialyzed overnight at 4°C against 50 mM Tris-HCl buffer 0
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191 containing 20% glycerol (pH 7.4). The protein concentrations were determined by the y

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192 Bradford method. The purified proteins were stored at -80°C. e
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193 Enzyme activity assays. The PAH activity was assayed using the method of
194 Bailey and Ayling (52) with minor modifications. The reaction mixture for PAH
195 contained 100 mM Tris-HCl (pH 7.0), 1 mM phenylalanine, 1 mg/ml catalase, and
196 0.14 µg/ml purified PAH-1 protein or 0.22 µg/ml purified PAH-2 protein, and was
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197 maintained at 25ºC for 5 min. Then, 100 µM ferrous ammonium sulphate was added
198 and allowed to incubate for 1 min. The reaction was started by the addition of 100 µM
199 BH4 (Sigma) or 6-methyltetrahydropterin (Sigma) and 5 mM DTT in a total volume of
200 50 µl. After a period of 15 min, the reaction was stopped by adding 50 µl 2 M
201 trichloroacetic acid. Samples were prepared for high performance liquid D
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202 chromatography (HPLC) after centrifugation. The HPLC was performed on a n
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203 SHIMADZU Model LC-20AT equipped with a Rheodyne loop of 20 µl, an Inertsil d
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204 ODS-3 column (5 µm, 150 x 4.6 mm; GL Science), and a SHIMADZU Model ro
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205 RF-20A fluorescence detector. The mobile phase consisted 0.1 M NH4OH adjusted to ht
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206 pH 4.6 with acetic acid at a flow rate of 1.5 ml/min. To observe the separation of //
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207 phenylalanine and tyrosine, the excitation and emission wavelengths of fluorescence .
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208 detector were set at 260 and 282 nm, respectively (53). To determine the amount of
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209 tyrosine, the excitation and emission wavelengths of fluorescence detector were set at /

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210 274 and 304 nm, respectively (52). Quantitation was based on external calibration A
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211 using a standard curve prepared with different amounts of tyrosine. il 4
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212 The reaction mixture for DHPR contained 100 mM Tris-HCl (pH 7.0), 10 µg/ml 0
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213 peroxidase, 10 mM hydrogen peroxide, 10 µM BH4, and 0.1 mM NADH. After an y

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214 incubation period of 4.5 min at 25 ºC, the reaction was started by adding 3.99 µg/ml e
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215 purified DHPR protein in a final volume of 1 ml. The initial rates were obtained from
216 the initial rate of decrease of A340 nm (e340 nm for NADH is 6200 M-1cm-1) (54, 55). The
217 background reactions were eliminated when the reaction was catalyzed by peroxidase
218 or DHPR alone.
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