Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.

The 2.0 A crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals a fold similar to that of tyrosine hydroxylase. It provides the first structural view of where mutations occur and a rationale to explain molecular mechanisms of the enzymatic phenotypes in the autosomal recessive disorder phenylketoneuria.

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Abstract. The events in the biogenesis of secretory granules after the budding of a dense-cored vesicle from the trans-Golgi network (TGN) were investigated in the neuroendocrine cell line PC12, using sulfatelabeled secretogranin II as a marker. The TGN-derived dense-cored vesicles, which we refer to as immature secretory granules, were found to be obligatory organellar intermediates in the biogenesis of the mature secretory granules which accumulate in the cell . Immature secretory granules were converted to mature secretory granules with a half-time of =45 min . This conversion entailed an increase in their size, implying that the maturation of secretory granules includes a fusion event involving immature secretory granules. Pulse-chase labeling of PC12 cells followed by stimulation with high T

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the ratelimiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118 -452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiledcoil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.Mammalian phenylalanine hydroxylase (PheOH, 1 phenylalanine 4-monooxygenase, EC 1.14.16.1) is an iron-and tetrahydropterin-dependent enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine. The reaction is the rate-limiting step in the catabolic pathway of phenylalanine resulting in the complete degradation of the amino acid.PheOH activity is tightly regulated by reversible phosphorylation and substrate activation. Mutations in the human PheOH gene result in the metabolic disorder known as phenylketonuria (PKU), a relatively common autosomal recessive disease that, if not diagnosed and properly treated from birth, ultimately leads to severe mental retardation. Approximately 280 mutant alleles of the human PheOH gene have been identified in patients with PKU and non-PKU hyperphenylalaninemia (1). The mutations lead to a variety of clinical and biochemical phenotypes with different degrees of severity (2, 3).PheOH shares many biochemical and physical properties with tyrosine hydroxylase (TyrOH) and tryptophan hydroxylase (TrpOH) suggesting that they evolved from a common ancestor and might constitute a family of structurally related proteins (for review, see Ref. 4). PheOH, TyrOH, and TrpOH catalyze similar hydroxylation reactions using the same nonheme iron atom but differ in their substrate specificity. All three hydroxylases are organized into three domains: a 12-19-kDa N-terminal domain involved in regulation of the activity followed by a segment of about 38 kDa, which consist of a catalytic domain and a 5-kDa tetramerization domain. The crystal structure of a dimeric form of PheOH (5) containing only the catalytic domain shows a remarkable similarity to the pr...