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Transformation of isopropylamine to L-alaninol by Pseudomonas sp. strain KIE171 involves N-glutamylated intermediates.

ABSTRACT: Pseudomonas sp. strain KIE171 was able to grow with isopropylamine or L-alaninol [S-(+)-2-amino-1-propanol] as the sole carbon source, but not with D-alaninol. To investigate the hypothesis that L-alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn5 mutants unable to utilize both isopropylamine and L-alaninol were isolated. Whereas mutant KIE171-BI transformed isopropylamine to L-alaninol, mutant KIE171-BII failed to do so. The two genes containing a transposon insertion were cloned, and the DNA regions flanking the insertions were sequenced. Two clusters, one comprising eight ipu (isopropylamine utilization) genes (ipuABCDEFGH) and the other encompassing two genes (ipuI and orf259), were identified. Comparisons of sequences of the deduced Ipu proteins and those in the database suggested that isopropylamine is transported into the cytoplasm by a putative permease, IpuG. The next step, the formation of gamma-glutamyl-isopropylamide from isopropylamine, ATP, and L-glutamate, was shown to be catalyzed by IpuC, a gamma-glutamylamide synthetase. gamma-Glutamyl-isopropylamide is then subjected to stereospecific monooxygenation by the hypothetical four-component system IpuABDE, thereby yielding gamma-glutamyl-L-alaninol [gamma(L-glutamyl)-L-hydroxy-isopropylamide]. Enzymatic hydrolysis by a hydrolase, IpuF, was shown to finally liberate L-alaninol and to regenerate L-glutamate. No gene(s) encoding an enzyme for the next step in the degradation of isopropylamine was found in the ipu clusters. Presumably, L-alaninol is oxidized by an alcohol dehydrogenase to yield L-2-aminopropionaldehyde or it is deaminated by an ammonia lyase to propionaldehyde. Genetic evidence indicated that the aldehyde formed is then further oxidized by the hypothetical aldehyde dehydrogenases IpuI and IpuH to either L-alanine or propionic acid, compounds which can be processed by reactions of the intermediary metabolism.

SUBMITTER: de Azevedo Wasch SI

PROVIDER: S-EPMC127573 | biostudies-literature | 2002 May

REPOSITORIES: biostudies-literature

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Transformation of isopropylamine to L-alaninol by Pseudomonas sp. strain KIE171 involves N-glutamylated intermediates.

de Azevedo Wäsch Susana I SI van der Ploeg Jan R JR Maire Tere T Lebreton Alice A Kiener Andreas A Leisinger Thomas T

Applied and environmental microbiology 20020501 5

Pseudomonas sp. strain KIE171 was able to grow with isopropylamine or L-alaninol [S-(+)-2-amino-1-propanol] as the sole carbon source, but not with D-alaninol. To investigate the hypothesis that L-alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn5 mutants unable to utilize both isopropylamine and L-alaninol were isolated. Whereas mutant KIE171-BI transformed isopropylamine to L-alaninol, mutant KIE171-BII failed to do so. The two genes containing a transposon insertio ...[more]

PMID: 11976110

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Project description:Nicotine and nicotinic acid (NA) are both considered to be representatives of N-heterocyclic aromatic compounds, and their degradation pathways have been revealed in Pseudomonas species. However, the cooccurrence of these two pathways has only been observed in Pseudomonas sp. strain JY-Q. The nicotine pyrrolidine catabolism pathway of strain JY-Q consists of the functional modules Nic1, Spm, and Nic2. The module enzyme, 3-succinoylpyridine monooxygenase (Spm), catalyzes transformation of 3-succinoyl-pyridine (SP) to 6-hydroxy-3-succinoyl-pyridine (HSP). There exist two homologous but not identical Spm enzymes (namely, Spm1 and Spm2) in JY-Q. However, when spm1 and spm2 were both in-frame deleted, the mutant still grew well in basic salt medium (BSM) supplemented with nicotine as the sole carbon/nitrogen nutrition, suggesting that there exists an alternative pathway responsible for SP catabolism in JY-Q. NicAB, an enzyme accounting for NA hydroxylation, contains reorganized domains similar to those of Spm. When the JY-Q_nicAB gene (nicAB in strain JY-Q) was introduced into another Pseudomonas strain, one that is unable to degrade NA, the resultant recombinant strain exhibited the ability to transform SP to HSP, but without the ability to metabolize NA. Here, we conclude that NicAB in strain JY-Q exhibits an additional role in SP transformation. The other genes in the NA cluster, NicXDFE (Nic2 homolog), then also exhibit a role in subsequent HSP metabolism for energy yield. This finding also suggests that the cooccurrence of nicotine and NA degradation genes in strain JY-Q represents an advantage for JY-Q, making it more effective and flexible for the degradation of nicotine.IMPORTANCE 3-Succinoyl-pyridine (SP) and 6-hydroxy-3-succinoyl-pyridine (HSP) are both valuable chemical precursors to produce insecticides and hypotensive agents. SP and HSP could be renewable through the nicotine microbial degradation pathway, in which 3-succinoylpyridine monooxygenases (Spm) account for transforming SP into HSP in Pseudomonas sp. strain JY-Q. However, when two homologous Spm genes (spm1 and spm2) were knocked out, the mutant retained the ability to degrade nicotine. Thus, in addition to Spm, JY-Q should have an alternative pathway for SP conversion. In this research, we showed that JY-Q_NicAB was responsible for this alternative SP conversion. Both of the primary functions for nicotinic acid dehydrogenation and the additional function for SP metabolism were detected in a recombinant strain harboring JY-Q_NicAB. As a result, both nicotinic acid and nicotine degradation pathways in JY-Q contribute to its remarkable nicotine tolerance and nicotine degradation availability. These findings also provide one more metabolic engineering strategy for accumulation for value-added intermediates.

Dataset Information

Transformation of isopropylamine to L-alaninol by Pseudomonas sp. strain KIE171 involves N-glutamylated intermediates.

Publications

Transformation of isopropylamine to L-alaninol by Pseudomonas sp. strain KIE171 involves N-glutamylated intermediates.

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