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Structure and Function of Amino Acid Ammonia-Lyases

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Structure and Function of Amino Acid Ammonia-Lyases Biocatalysis and Biotransformation, 2004 VOL. 22 (2). pp. 131Á/138 Structure and Function of Amino Acid Ammonia-lyases YASUHISA ASANOa*, YASUO KATOa, COLIN LEVYb, PATRICK BAKERb and DAVID RICEb a Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398 Japan; b Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, S10 2TN United Kingdom Histidine ammonia-lyase (HAL) and methylaspartate elimination involving abstraction of a non-acidic and ammonia-lyase (MAL) belong to the family of carbon- inactive b hydrogen (Walsh, 1979). nitrogen lyases (EC 4.3.1). The enzymes catalyze the a,b-elimination of ammonia from (S)-His to yield uro- In 1960, a Japanese company Tanabe Seiyaku canic acid, and (S)-threo-(2S,3S)-3-methylaspartic acid to started the industrial production of (S)-Asp from mesaconic acid, respectively. Based on structural ana- fumaric acid by immobilized cells of Escherichia coli lyses, the peptide at the active center of HAL containing AAL as one of the examples of the from Pseudomonas putida is considered to be post- industrial use of the enzyme (van der Werf et al., translationally dehydrated to form an electrophilic 4-methylidene-imidazole-one (MIO) group. A reaction 1994). It has also been discovered by Japanese mechanism was proposed with the structure. On the researchers that PAL catalyzes not only the degrada- other hand, the structure of MAL from Citrobacter tion of (S)-Phe, but also the synthesis of (S)-Phe from amalonaticus was found to be a typical TIM barrel 2 trans-cinnamic acid in a high concentration of structure with Mg coordinated to the 4-carbonyl of the ammonia up to 5 M (Yamada et al., 1981). substrate methylaspartate. Unlike HAL, MIO was not observed in MAL, and the reaction of MAL appears to be In this review, we outline how the structures have completely different from phenylalanine ammonia-lyase been elucidated and the possible reaction mechan- (PAL), HAL, and other amino acid ammonia-lyases. A isms of amino acid ammonia-lyases studied by the reaction mechanism is proposed in which the hydrogen groups of Re´tey in Germany, and Rice in England at the b to the amino group of the substrate is abstracted and ourselves. There are excellent recent reviews on forming an enolate type intermediate and then ammonia is released. HAL and PAL by Re´tey et al. (Langer et al., 2001; Re´tey, 2003), and AAL by Viola (2000). Keywords: Amino acid ammonia-lyase; Histidine ammonia-lyase; 3-Methylaspartate ammonia-lyase; X-ray crystallography HISTIDINE AMMONIA-LYASE (HAL) AND PHENYLALANINE AMMONIA-LYASE (PAL) HAL catalyzes the degradation of (S)-His to urocai- INTRODUCTION nic acid and ammonia, and has been purified from Pseudomonas sp. and other biological sources. Ables A group of enzymes called amino acid ammonia- et al., have shown that HAL may have an active and lyases has intrigued enzymologists and organic essential electrophilic group at the active site be- chemists, since the reactions cannot be done non- cause it was inactivated by NaBH4, but also a enzymatically, and the enzymes catalyze the addi- carbonyl group, because the enzyme was inactivated tion of ammonia to achiral olefinic acids to form by nucleophiles such as phenylhydrazine, NaHSO3, chiral L-amino acids (Hanson and Havir, 1973). NH2OH, and KCN (Smith et al., 1967). Later, they These enzymes include histidine ammonia-lyase inactivated HAL with [14C]-nitromethane and re- (HAL; EC 4.3.1.3), phenylalanine ammonia-lyase duced with NaBH4, and found that the radioactivity (PAL; EC 4.3.1.5), aspartate ammonia-lyase (AAL; was incorporated into 4-amino-2-hydroxybutyric EC 4.3.1.2), and 3-methylaspartate ammonia-lyase acid, 2,4-diaminobutyric acid and b-alanine after (3-methylaspartase, MAL; EC 4.3.1.1), etc. These hydrolysis. They considered that the electrophilic enzymes pose a mechanistic challenge as to how group of HAL could be dehydroalanine and the they remove ammonia from L-amino acids by trans amino group of (S)-His may form a Schiff base with * Corresponding author. Tel.: /81-766-56-7500. Fax: /81-766-56-2498. E-mail: [email protected] ISSN 1024-2422 print/ISSN 1029-2446 online # 2004 Taylor & Francis Ltd DOI: 10.1080/10242420410001703496 132 Y. ASANO et al. an unidentified carbonyl group (Givot et al., 1969). been reported to occur in the peptide lantiobiotics This hypothesis was also supported by the fact that such as Nisin (Sahl et al., 1995). (S)-His prevented the inactivation and 0.9 mol Comparison of the primary structures of HAL and [3H]Ala per mole of the enzyme was obtained, PAL from various sources showed that four Ser when HAL was inactivated and reduced with residues are conserved (Taylor et al., 1991). When the 3 NaB H4 and then hydrolyzed (Wickner, 1969). conserved Ser residues were changed to Ala by site- A similar observation was obtained with PAL, directed mutagenesis, only the Ser143Ala mutant 3 14 when it was inactivated with NaB H4 or [ C]cya- was inactivated, therefore Ser143 was considered to nide and then hydrolyzed with HCl and subjected be the precursor of the prosthetic group in HAL and to amino acid analysis. The radioactivity was present Ser202 in PAL (Langer et al., 1994). Furthermore, the 14 in Ala or Asp, respectively. Four moles of K CN sequence containing the active Ser, GlyÁ/SerÁ/Val Á/ per mole of the tetrameric enzyme was incor- GlyÁ/AlaÁ/SerÁ/GlyÁ/AspÁ/LeuÁ/AlaÁ/ProÁ/Leu (con- porated, and with an intermediate formation of served amino acid residues are shown underlined) b-cyanoalanine, [14C]Asp was obtained as a product was highly conserved in most of HAL and PAL (Consevage and Philips, 1985; Hodgins, 1971) (Poppe, 2001). Since Ser202 in Parsley PAL was Hanson and Havir (1970) have postulated a possible considered to be the precursor of the prosthetic reaction mechanism of HAL and PAL (Fig. 1), in group (Schuster and Re´tey, 1995), the Ser was which the initiation step is a nucleophilic attack of changed to Thr and Gly with the loss of most of the substrate amino group on the electrophilic group the activity. Although it was not shown that the of the enzyme (dehydroalanyl residue). prosthetic group was dehydroalanine, it has been How might such an electrophilic prosthetic group strongly suggested the Ser residue is the precursor of function in the catalytic deamination reaction (Han- dehydroalanine (Langer et al., 1994). son and Havir, 1973; Walsh, 1979)? Attack of the substrate amino group at the b-carbon of the Structure of HAL and Discovery of MIO dehydroalanyl residue could occur in a net 1,4- addition step. To activate the initial complex for The structure of HAL from Pseudomonas putida was loss of the amino moiety, a 1,3-prototopic shift could solved to 1.8 A˚ resolution (Schwede et al., 1999). occur, producing an eneamine with the conjugated HAL is a homo tetrameric enzyme with 509 amino a,b-unsaturated carbonyl system. The enamine acts acids per peptide and is subdivided into two sub- as an electron sink to cleave the CÁ/N bond, and domains. The N terminus region (40%) is a globular trans-cinnamic acid is formed. Expulsion of ammo- domain with 8 helices and 4 short b sheets. The C nia could proceed together with regeneration of the terminal domain is composed of a core of 5 long initial dehydroalanyl residue at the active site by a parallel a helices surrounded by 6 further helices. 1,3-prototropic shift. Although there is no notable similarity between the primary structure of HAL and other ammonia- lyases, its tertiary structure resembles that of tetra- Origin of the Electrophilic Group meric lyases such as fumarase (Weaver et al., 1995), Dehydroalanine could be formed by the dehydration AAL (Fujii et al., 2003; Shi et al., 1997), arginosucci- of the precursor serine in the enzyme. It has also nate lyase (Turner et al., 1997). FIGURE 1 Proposed reaction mechanism of PAL with active site dehydroalanine (Hanson and Havir, 1970; Walsh, 1979). STRUCTURE AND FUNCTION OF AMINO ACID AMMONIA-LYASES 133 The most striking discovery in the X-ray crystal- lacking dehydroalanine and the NaBH4-treated en- lographic analysis of HAL is the existence of the zyme with 5-nitro-(S)-His as substrate ruled out the catalytically important electrophilic group, 4-meth- mechanism in which the a-amino group of (S)-His ylidene imidazole-1-one (MIO) (Fig. 2). MIO can be attacks this electrophilic group as proposed in Fig. 1. considered as a modified dehydroalanine with With these observations, together with the X-ray strong electrophilicity (Poppe, 2001; Re´tey, 2003). crystallographic analysis, a reaction mechanism for Interestingly, when various HAL genes were ex- HAL was proposed (Schwede et al., 1999) (Fig. 3). pressed in translation systems such as E. coli and When (S)-His is bound to the enzyme, the electron COS cells, the Ala142Á/Serl43Á/Gly144 residue was pair from the imidazole ring of (S)-His attacks the autocatalytically modified to MIO. It is considered methylidene group of MIO. The cation formed at the that dehydration reactions occur after the exomethy- imidazole ring increases the acidity of the hydrogen lene structure of MIO is formed by a cyclization atom at the b position of (S)-His. A basic group in reaction (Baedeker and Schulz, 2002) (Fig. 2). MIO the enzyme abstracts the proton at the b position has a maximum absorption at 308 nm and shoulder forming urocanic acid and ammonia, regenerating at 315 nm. Although the X-ray structure analysis of an electrophilic prosthetic group.
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