Genes and Enzymes of Azetidine-2-Carboxylate Metabolism: Detoxification and Assimilation of an Antibiotic

Abstract

L-(؊)-Azetidine-2-carboxylate (AC) is a toxic, natural product analog of L-proline. This study revealed the genes and biochemical strategy employed by Pseudomonas sp. strain A2C to detoxify and assimilate AC as its sole nitrogen source. The gene region from Pseudomonas sp. strain A2C required for detoxification was cloned into Escherichia coli and sequenced. The 7.0-kb region contained eight identifiable genes. Four encoded putative transporters or permeases for ␥-amino acids or drugs. Another gene encoded a homolog of 2-haloacid dehalogenase (HAD). The encoded protein, denoted L-azetidine-2-carboxylate hydrolase (AC hydrolase), was highly overexpressed by subcloning. The AC hydrolase was shown to catalyze azetidine ring opening with the production of 2-hydroxy-4-aminobutyrate. AC hydrolase was further demonstrated to be a new hydrolytic member of the HAD superfamily by showing loss of activity upon changing aspartate-12, the conserved active site nucleophile in this family, to an alanine residue. The presence of a gene encoding a potential export chaperone protein, CsaA, adjacent to the AC hydrolase gene suggested that AC hydrolase might be found inside the periplasm in the native Pseudomonas strain. Periplasmic and cytoplasmic cell fractions from Pseudomonas sp. strain A2C were prepared. A higher specific activity for AC hydrolysis was found in the periplasmic fraction. Protein mass spectrometry further identified AC hydrolase and known periplasmic marker proteins in the periplasmic fraction. A model was proposed in which AC is hydrolyzed in the periplasm and the product of that reaction is transported into and further metabolized in the cytoplasm.Plants produce many toxins that offer protection against grazing by insects and mammals (11). Some of those toxins act by mimicking essential amino acids. One such natural product is S-(Ϫ)-azetidine-2-carboxylate (AC), which can comprise up to 7% of the leaf mass of Lily of the Valley plants (6, 7) and has more recently been found to be present in sugar beets (26). AC mimics the amino acid L-proline. Upon ingestion by a susceptible animal, AC becomes incorporated into proteins in place of L-proline. The different bond angles in AC compared to L-proline causes change in the protein backbone, which can result in disruption of function (8). AC is also produced by Streptomyces spp. and is considered to be an antibiotic, but it is too toxic to humans to be useful clinically.AC is toxic to microorganisms if it is transported into the cell and incorporated into proteins (8). However, Saccharomyces cerevisiae is known to detoxify AC via acetylation of the carboxylic acid group (32). Additionally, bacteria are known that not only tolerate AC but use it as the sole source of nitrogen (5,35,36). Bacteria growing on AC are reported to open the azetidine ring and susbsequently capture the nitrogen atom via a transamination reaction. The data are consistent with a mechanism involving an enzyme-catalyzed hydrolytic opening of the azetidine ring. However, until the present study, th...

Crystallization and preliminary X-ray analysis ofL-azetidine-2-carboxylate hydrolase fromPseudomonassp. strain A2C

“…The recombinant plasmid pET29-A2CH was constructed as reported previously (Gross et al, 2008). Escherichia coli strain BLR (DE3) (Novagen, Madison, Wisconsin, USA) was transformed with the plasmid for A2CH overexpression.…”

“…A2CH showed no enzymatic activity with either (R)-or (S)-2chloropropionic acid (Gross et al, 2008). While some important residues in l-DEX are conserved in A2CH, such as the aspartate nucleophile, other important residues are not.…”

“…l-Azetidine-2-carboxylate hydrolase (A2CH) from Pseudomonas sp. strain A2C catalyzes the hydrolytic opening of the azetidine ring of (S)-azetidine-2-carboxylic acid (AZC) and generates the product 2-hydroxy-4-aminobutyric acid (Gross et al, 2008). AZC, the substrate of the enzyme, is an analogue of l-proline and is biosynthesized by some plants such as lily of the valley.…”

Methods of Biotechnology in the Synthesis of Drugs (2nd Ed.)

“…Восстановление α-кето-β-лактама (CCIV) растущими клетками дрожжей Saccharomyces cerevisiae приводило в течение пяти суток к образованию цисгидроксипроизводного (CCV, выход 62%) и трансгидроксипроизводного (CCVI, выход 38%) [Mihovilovic et al, 2005 Превращение азетидин-2-карбоновой кислоты (CCVII) гидролазой Pseudomonas sp. strain A2C сопровождалось образованием 2-гидрокси-4аминомасляной кислоты (CCVIII) [Gross et al, 2008]:…”

10.17686/sced_rusnauka_2014-1382

“…Превращение азетидин-2-карбоновой кислоты (CCVII) гидролазой Pseudomonas sp. strain A2C сопровождалось образованием 2-гидрокси-4-аминомасляной кислоты (CCVIII) (Gross et al, 2008):…”