SUMMARYGrowing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate-limiting enzymes and negative feedback inhibition by end-products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)-norcoclaurine-6-O-methyltransferase (6OMT), a key rate-limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end-products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S-adenosyl-L-homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time-dependent sensitivity toward sanguinarine.

Edited by Ulf-Ingo Flügge Keywords:Aspartate aminotransferase Aromatic amino acid Enzymology Metabolism Plant Prephenate aminotransferase a b s t r a c tIn all organisms synthesising phenylalanine and/or tyrosine via arogenate, a prephenate aminotransferase is required for the transamination of prephenate into arogenate. The identity of the gene encoding this enzyme in the organisms where this activity occurs is still unknown. Glutamate/aspartate-prephenate aminotransferase (PAT) is thus the last homeless enzyme in the aromatic amino acids pathway. We report on the purification, mass spectrometry identification and biochemical characterization of Arabidopsis thaliana prephenate aminotransferase. Our data revealed that this activity is housed by the prokaryotic-type plastidic aspartate aminotransferase (At2g22250). This represents the first identification of a gene encoding PAT.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) intohomogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme. 4-Hydroxyphenylpyruvate dioxygenase (HPPD)3 is an Fe II -dependent non-heme oxygenase catalyzing the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate (HGA). In most aerobic life forms, HPPD catalyzes the second step in tyrosine catabolism. In all photosynthetic organisms, HPPD also plays an anabolic role, as HGA is essential for the formation of isoprenoid redox molecules such as plastoquinone and tocochromanols (1, 2). Plant HPPD is the molecular target of several natural compounds (3, 4) and of a range of very effective synthetic herbicides that are currently used commercially (5-9). In mammals, inborn defects in this pathway give rise to metabolic disorders of different degrees of severity (5, 10). Among these, two involve HPPD. Type III tyrosinemia arises from low HPPD activity (11) caused by an alanine to valine mutation at position 268 in the human enzyme (12). Hawkinsinuria, linked with an active enzyme with almost uncoupled turnover, is a result of an alanine to threonine mutation at position 33 in the human enzyme (12). This mutant enzyme releases an arene oxide-derived intermediate excreted in large quantities in the urine (13).Interestingly, HPPD inhibitor/herbicide molecules also act as therapeutic agents for the debilitating and lethal inborn defects associated with type I tyrosinemia. Inhibition of HPPD prevents the accumulation of toxic metabolites in this disease (5). HPPD inhibitors are also currently being used in trials for treatment of Parkinson disease, based on the premise that inhibition of tyrosine catabolism will increase tyrosine availability for conversion to 3,4-dihydroxyphenylalanine in the brain.The reaction mechanism of HPPD is complex; it first involves the nucleophilic attack of the ␣-keto ...

The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1β in tested α- and β-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the β-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.

Alternative routes for the post‐chorismate branch of the biosynthetic pathway leading to tyrosine exist, the 4‐hydroxyphenylpyruvate or the arogenate route. The arogenate route involves the transamination of prephenate into arogenate. In a previous study, we found that, depending on the microorganisms possessing the arogenate route, three different aminotransferases evolved to perform prephenate transamination, that is, 1β aspartate aminotransferase (1β AAT), N‐succinyl‐l,l‐diaminopimelate aminotransferase, and branched‐chain aminotransferase. The present work aimed at identifying molecular determinant(s) of 1β AAT prephenate aminotransferase (PAT) activity. To that purpose, we conducted X‐ray crystal structure analysis of two PAT competent 1β AAT from Arabidopsis thaliana and Rhizobium meliloti and one PAT incompetent 1β AAT from R. meliloti. This structural analysis supported by site‐directed mutagenesis, modeling, and molecular dynamics simulations allowed us to identify a molecular determinant of PAT activity in the flexible N‐terminal loop of 1β AAT. Our data reveal that a Lys/Arg/Gln residue in position 12 in the sequence (numbering according to Thermus thermophilus 1β AAT), present only in PAT competent enzymes, could interact with the 4‐hydroxyl group of the prephenate substrate, and thus may have a central role in the acquisition of PAT activity by 1β AAT.

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