Heterologous Expression and Characterization of Phenylalanine Hydroxylase from Chromobacterium violaceum

Abstract

Abstract:

Phenylalanine hydroxylase（PAH）derived from Chromobacterium violaceum has potential pharmaceutical value due to its simple structure and the closer property to human PAH. We cloned the pah gene from C. violaceum and constructed recombinant expression vcectors pET24a-pah, finally, the pah gene was efficiently expressed in Escherichia coli BL21（DE3）. The specific activity of recombination protein was 503.2 U/mg after purification. The studies of enzymatic properties showed that the enzyme displayed maximum activity at 40℃, and its half-life was 15 min at 50℃. The optimal pH was pH7.5, and the enzyme activity was stable at the range of pH6-8. Under the conditions of 37℃ and pH7.5, its K_m was 1.5 mmol/L, V_max was 0.5 mmol/min, k_cat was 5.05/s, and the catalytic efficiency（k_cat /K_m）was 3.37 L/mmol·s.

Key words: Phenylalanine hydroxylase, Chromobacterium violaceum, Expression, Purification, Enzymatic properties

Cao Shuhui, Zhou Li, Cui Wenjing, Liu Zhongmei, Zhou Zhemin. Heterologous Expression and Characterization of Phenylalanine Hydroxylase from Chromobacterium violaceum[J]. Biotechnology Bulletin, 2014, 0(8): 152-158.

References

［1］Ronau JA, Paul LN, Fuchs JE, et al. An additional substrate binding site in a bacterial phenylalanine hydroxylase［J］. Eur Biophys J, 2013, 42（9）：691-708.
［2］Erlandsen H, Kim JY, Patch MG, et al. Structural comparison of bcterial and human iron-dependent phenylalanine hydroxylases：similar fold, different stability and reaction rates［J］. J Mol Bio, 2002, 320（3）：645-661.
［3］Fitzpatrick PF. Mechanism of aromatic amino acid hydroxylation［J］. Biochemistry, 2003, 42（48）：14083-14091.
［4］王芳, 奚耕思, 刘丽萍, 等.苯丙氨酸羟化酶的研究进展［J］.生命科学, 2013, 25（4）：372-377.
［5］Loaiza A, Armstrong KM, Baker BM, et al. Kinetics of thermal unfolding of phenylalanine hydroxylase variants containing different metal cofactors（FeII, CoII, and ZnII）and their isokinetic relationship［J］. Inorg Chem, 2008, 47（11）：4877-4883.
［6］Chen D, Frey PA. Phenylalanine hydroxylase from Chromobac-terium violaceum uncoupled oxidation of tetrahydropterin and the role of iron in hyroxylation［J］. J Biol Chem, 1998, 273（40）：25594-25601.
［7］林琳, 张可, 钱和.辐照鸡肉邻位酪氨酸含量检测方法的研究［J］.食品科学, 2010, 31（10）：188-191.
［8］Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding［J］. Anal Biochem, 1976, 72（3）：248-254.
［9］Pember SO, Benkovic SJ, Villafranca JJ, et al. Adduct formation between the cupric site of phenylalanine hydroxylase from Chromob-acterium violaceum and 6, 7-Dimethyltetrahydropterin［J］. Bioche-mistry, 1987, 26（14）：4477-4483.
［10］Nakata H, Yamauchi T, Fujisawa H. Phenylalanine hydroxylase from Chromobacterium violaceum purification and characterization［J］. J Biol Chem, 1979, 254（6）：1829-1833.
［11］Pember SO, Villafranca JJ, Benkovic SJ. Phenylalanine hydroxylase from Chromobacterium violaceum is a copper-containing monooxy-genase. kinetics of the reductive activation of the enzyme［J］. Biochemistry, 1986, 25（21）：6611-6619.
［12］Onishi A, Liotta LJ, Benkovic SJ. Cloning and expression of Chro-mobacterium violaceurn phenylalanine hydroxylase in Escherichia coli and comparison of amino acid sequence with mammalian aromatic amino acid hydroxylas［J］. J Biol Chem, 1991, 266（28）：18454-18459.
［13］Koehntop KD, Emerson JP, Que L Jr. The 2-his-1-carboxylate facial triad：a versatile platform for dioxygen activation by mononuclear non-heme iron（II）enzymes［J］. J Biol Inorg Chem, 2005, 10（2）：87-93.
［14］Yew NS, Dufour E, Przybylska M, et al. Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice［J］. Mol Genet Metab, 2013, 109（4）：339-344.

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