Crystal Structure Analysis of Free and Substrate-Bound 6-Hydroxy-l-Nicotine Oxidase from Arthrobacter nicotinovorans

Качалова, Г.С.; Bourenkov, Gleb; Mengesdorf, Thorsten; Schenk, Susann; Maun, Henry R.; Burghammer, Manfred; Riekel, Christian; Decker, Karl; Bartunik, H.D.

doi:10.1016/j.jmb.2009.12.009

Cited by 33 publications

(42 citation statements)

References 46 publications

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“…The GϩC content was 50.67%. Compared with the known enzymes available from the Protein Data Bank (PDB, NCBI database), the predicted protein showed the highest similarity with several flavin-containing amine oxidases, e.g., the 6-hydroxy-Lnicotine oxidase (6HLNO) from Arthrobacter nicotinovorans (29% identity) (11,12), the monoamine oxidase from Aspergillus niger (28% identity) (1), the human monoamine oxidase A (23% identity) (9), L-amino acid oxidase from Calloselasma rhodostoma (22% identity) (13), and the polyamine oxidase Fms1 from Saccharomyces cerevisiae (24% identity) (15).…”

Section: Resultsmentioning

confidence: 99%

“…The 6HLNO also uses a noncovalent FAD as the cofactor. According to the amino acid sequence alignment results, the most similar regions between PNAO and 6HLNO were the three FAD binding domains, which are the residues 1 to 81, 200 to 285, and 361 to 425 of 6HLNO (12) and the corresponding residues 59 to 135, 258 to 344, and 425 to 489 of PNAO, respectively. Although they shared 29% sequence identity with one another, the enzymatic reactions catalyzed by 6HLNO and PNAO were distinct.…”

Section: Discussionmentioning

confidence: 99%

“…6D). The first enzymatic step was catalyzed by an amine oxidase, similar to the step catalyzed by 6HLNO (11,12), converting nicotine to N-methylmyosmine and spontaneously yielding PN. The second and third enzymatic steps were catalyzed by PNAO and SAPD, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…In the Gram-positive strain Arthrobacter nicotinovorans, the degradation pathway (the pyridine pathway) and the related genes and enzymes have been well elucidated (2,(5)(6)(7)(10)(11)(12). Although aerobic nicotine degradation has been studied in different Gram-negative Pseudomonas strains, the genes encoding enzymes in the pyrrolidine pathway involved in nicotine degradation have yet to be analyzed in detail (16).…”

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confidence: 99%

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Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of Nicotine

Qiu

Wen

et al. 2012

Appl Environ Microbiol

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Nicotine is a natural alkaloid produced by tobacco plants, and the mechanisms of its catabolism by microorganisms are diverse. In the present study, we reported the mutation, cloning, and identification of two novel genes involved in nicotine degradation from the newly isolated Pseudomonas sp. strain HZN6. Transposon mutagenesis identified a HZN6 mutant in which the nicotine-degrading pathway was blocked at pseudooxynicotine. A 3,874-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR. Two open reading frames (designated pao and sap) were analyzed, and the deduced amino acid sequences shared 29% identity with 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans and 49% identity with an aldehyde dehydrogenase from Bartonella henselae. Both pao and sap were cloned and functionally expressed in recombinant Escherichia coli BL21. The pao gene encoded a novel pseudooxynicotine amine oxidase with noncovalently bound flavin adenine dinucleotide (FAD) and exhibited substrate specificity removing the methylamine from pseudooxynicotine with the formation of 3-succinoylsemialdehyde-pyridine and hydrogen dioxide. The sap gene encoded a NADP ؉ -dependent 3-succinoylsemialdehyde-pyridine dehydrogenase that catalyzed the dehydrogenation of 3-succinoylsemialdehyde-pyridine to 3-succinoyl-pyridine. Genetic analyses indicated that the pao gene played an essential role in nicotine or pseudooxynicotine mineralization in strain HZN6, whereas the sap gene did not. This study provides novel insight into the nicotine-degrading mechanism at the genetic level in Pseudomonas spp.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of Nicotine

Qiu

Wen

et al. 2012

Appl Environ Microbiol

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“…Nicotine is a naturally chiral compound, and the enantiomeric (S-isomer/R-isomer) ratio is approximately 200:1 (40). The biodegradation of (RS)-nicotine in the strain A. nicotinovorans showed chiral selectivity that has been well elucidated (16,40,41). However, selective degradation of (RS)-nicotine in Pseudomonas strains has been poorly studied.…”

Section: Discussionmentioning

confidence: 99%

Cloning of a Novel Nicotine Oxidase Gene from Pseudomonas sp. Strain HZN6 Whose Product Nonenantioselectively Degrades Nicotine to Pseudooxynicotine

Qiu

Zhang

et al. 2013

Appl Environ Microbiol

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bPseudomonas sp. strain HZN6 utilizes nicotine as its sole source of carbon, nitrogen, and energy. However, its catabolic mechanism has not been elucidated. In this study, self-formed adaptor PCR was performed to amplify the upstream sequence of the pseudooxynicotine amine oxidase gene. A 1,437-bp open reading frame (designated nox) was found to encode a nicotine oxidase (NOX) that shows 30% amino acid sequence identity with 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans. The nox gene was cloned into a broad-host-range cloning vector and transferred into the non-nicotine-degrading bacteria Escherichia coli DH5␣ (DH-nox) and Pseudomonas putida KT2440 (KT-nox). The transconjugant KT-nox obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine, while DH-nox did not. Reverse transcription-PCR showed that the nox gene is expressed in both DH5␣ and KT2440, suggesting that additional factors required for nicotine degradation are present in a Pseudomonas strain(s), but not in E. coli. The mutant of strain HZN6 with nox disrupted lost the ability to degrade nicotine, but not pseudooxynicotine. These results suggested that the nox gene is responsible for the first step of nicotine degradation. The (RS)-nicotine degradation results showed that the two enantiomers were degraded at approximately the same rate, indicating that NOX does not show chiral selectivity. Site-directed mutagenesis revealed that both the conserved flavin adenine dinucleotide (FAD)-binding GXGXXG motif and His456 are essential for nicotine degradation activity.

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Development of an R‐Selective Amine Oxidase with Broad Substrate Specificity and High Enantioselectivity

et al. 2014

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Amine oxidases are useful bio‐catalysts for the synthesis of enantiomerically pure 1°, 2° and 3° chiral amines. Enzymes in this class (e.g., MAO‐N from Aspergillus niger) reported previously have been shown to be highly S selective. Herein we report the development of an enantiocomplementary R‐selective amine oxidase based on 6‐hydroxy‐D‐nicotine oxidase (6‐HDNO) with broadened substrate scope and high enantioselectivity. The engineered 6‐HDNO enzyme has been applied to the preparative deracemisation of a range of racemic amines to yield S‐configured products, for example, (S)‐nicotine, in high ee.

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Crystal Structure Analysis of Free and Substrate-Bound 6-Hydroxy-l-Nicotine Oxidase from Arthrobacter nicotinovorans

Cited by 33 publications

References 46 publications

Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of Nicotine

Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of Nicotine

Cloning of a Novel Nicotine Oxidase Gene from Pseudomonas sp. Strain HZN6 Whose Product Nonenantioselectively Degrades Nicotine to Pseudooxynicotine

Development of an R‐Selective Amine Oxidase with Broad Substrate Specificity and High Enantioselectivity

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