Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540

Hellemond, Erik W. van; Dijk, Marianne van; Heuts, Dominic P. H. M.; Janssen, Dick B.; Fraaije, Marco W.

doi:10.1007/s00253-007-1310-4

Cited by 38 publications

(72 citation statements)

References 31 publications

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“…ORF4 was designated styA1, since the deduced protein showed a high level of similarity to a large number of oxygenase subunits of putative two-component SMOs. The very few among them whose activity was experimentally proven are SMOs from pseudomonads (e.g., the main component, StyA, of Pseudomonas fluorescens strain ST [4]) or from an uncultured bacterium (subunit A [61]) (Table 2). ORF5, which is cotranscribed and located directly downstream of ORF4, was found to be most remarkable, since the encoded protein seems to represent a fusion between an oxygenase and a flavin oxidoreductase.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

et al. 2009

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Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His 10 -StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.The incorporation of one atom of oxygen during hydroxylation, epoxidation, sulfoxidation, or Baeyer-Villiger oxidation is a common initial step of the aerobic degradation of aromatic compounds by microorganisms. In bacteria, these reactions are most frequently catalyzed by inducible flavoprotein monooxygenases (EC 1.14.13 [57]). The majority of these enzymes (socalled single-component flavoprotein monooxygenases) utilize electrons from NAD(P)H, which are transferred to a noncovalently bound flavin adenine dinucleotide (FAD) in order to activate molecular oxygen as a flavin (hydro)peroxide. Depending on the protonation of this intermediate and the type of substrate, an oxygen atom is then incorporated by nucleophilic or electrophilic attack. More recently, different twocomponent flavoprotein monooxygenases have been characterized (57). These systems cover an NAD(P)H-dependent flavin reductase in order to generate reduced flavin and an oxygenase that utilizes this cofactor for the activation of oxygen.The exquisite regio-and stereoselectivities of oxygen insertion by flavoprotein monooxygenases favor these enzymes for biocatalytic applications (23,24,33). This is especially true because chemical synthesis approaches by hetero-or homogenic catalysis often do not yield a sufficiently high enantiomeric excess for the production of pharmaceuticals and their chiral building blocks. The use of oxygen as an inexpensive nontoxic oxidant and mild reaction conditions are additional advantages with the potential for increasing the environmental sustainability of oxygenase-catalyzed bio...

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Section: Resultsmentioning

confidence: 99%

Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

et al. 2009

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“…Expression and Purification of Wild-type PuO Rh and PuO Rh A394C-Expression of PuO Rh and PuO Rh A394C in E. coli TOP10 was performed as described before (11). For expression of PuO Rh A394C in E. coli ORIGAMI, these cells were transformed with pBAD-PuO Rh A394C and cultivated in TB medium supplemented with 50 g/ml ampicillin, 15 g/ml kanamycin, 12.5 g/ml tetracycline, and 0.4% (w/v) L-(ϩ)-arabinose at 30°C for 24 h. Purification of wild-type PuO Rh and PuO Rh A394C was performed as described before (11).…”

Section: Methodsmentioning

confidence: 99%

“…Measurement of PuO Rh A394C activity was done by using a coupled horseradish peroxidase assay as described before (11). Rh -To determine the exact molecular mass of wild-type PuO Rh , we analyzed the purified protein (2.0 M) by nanoflow ESI-MS.…”

Section: Methodsmentioning

confidence: 99%

“…The human oxidase shares 32% sequence identity with PuO Rh and catalyzes the oxidative deamination of aromatic monoamines like phenylethylamine and benzylamine. Based on the MAO-B structure we have constructed a structural model for PuO Rh (11). By inspection of the model and subsequent site-directed mutagenesis studies, we have verified that one specific active site residue (Glu-324) in PuO Rh is crucial for its preference for aliphatic diamine substrates.…”

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confidence: 88%

“…Recently we identified a novel putrescine oxidase from the actinomycete Rhodococcus erythropolis (PuO Rh ) (11). PuO Rh shares 67% sequence identity with PuO Mr and displays similar catalytic properties: it is highly specific for putrescine and is effectively inhibited by aliphatic monoamines and short diamines.…”

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ADP Competes with FAD Binding in Putrescine Oxidase

Hellemond

Mazon

Heck

et al. 2008

Journal of Biological Chemistry

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Putrescine oxidase (PuO, 4 EC 1.4.3.10) is a flavin-containing amine oxidase involved in polyamine degradation. It catalyzes the oxidative deamination of putrescine (1,4-diaminobutane) into 4-aminobutanal with concomitant reduction of oxygen to hydrogen peroxide (Scheme 1). In addition, the oxidase also accepts other polyamines, like spermine and spermidine, which are involved in cell growth and differentiation and interact with nucleic acids (1, 2).PuO was first reported in the 1960s when it was isolated from Micrococcus rubens (3, 4). This enzyme (PuO Mr ) is a soluble homodimeric protein and is unique among flavoprotein oxidases in that the isolated enzyme appears to contain only one noncovalently bound FAD molecule per dimeric protein (5). PuO Mr was heterologously expressed in Escherichia coli (6) and is applied as a diagnostic enzyme for the detection of di-and polyamines (7,8). Increased levels of polyamines in tissue and body fluids are related to cancer and thus can be used as tumor markers (9). PuO Mr can also be used to monitor food freshness by detecting polyamines (10).Recently we identified a novel putrescine oxidase from the actinomycete Rhodococcus erythropolis (PuO Rh ) (11). PuO Rh shares 67% sequence identity with PuO Mr and displays similar catalytic properties: it is highly specific for putrescine and is effectively inhibited by aliphatic monoamines and short diamines. Like PuO Mr , PuO Rh is a homodimer of about 100 kDa and, interestingly, also contains only one mol of noncovalently bound FAD per mol of dimeric protein. Such a low flavin cofactor occupancy is remarkable as inspection of the PuO sequences suggests that each monomer contains a Rossmanfold domain capable of FAD binding.To explain the reason of the unusually low FAD/protein ratio in PuO, more detailed structural information would be highly informative. Unfortunately, no crystal structure is available for a PuO. However, for the sequence-related human monoamine oxidase B (MAO-B), the crystal structure has been solved (12). MAO-B is a flavoprotein oxidase located at the outer mitochondrial membrane (13) and is involved in the oxidation of neurotransmitters (14). The human oxidase shares 32% sequence identity with PuO Rh and catalyzes the oxidative deamination of aromatic monoamines like phenylethylamine and benzylamine. Based on the MAO-B structure we have constructed a structural model for PuO Rh (11). By inspection of the model and subsequent site-directed mutagenesis studies, we have verified that one specific active site residue (Glu-324) in PuO Rh is crucial for its preference for aliphatic diamine substrates. Like PuO Mr and PuO Rh , MAO-B is a homodimeric protein, but in contrast to both bacterial oxidases, each MAO-B subunit contains one covalently bound FAD cofactor. The FAD in MAO-B is covalently attached to a cysteine residue (Cys-397) via a C8␣-linkage.In this report, we describe our findings concerning a more detailed analysis of cofactor binding in PuO Rh . The data reveal that ADP is competing with FAD binding. Fur...

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The repertoire of nitrogen assimilation in Rhodococcus: catalysis, pathways and relevance in biotechnology and bioremediation

Foster

Barnes

Speight

et al. 2014

J of Chemical Tech & Biotech

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The Rhodococcus genus exhibits diverse enzymatic activity that can be exploited in the conversion of natural and anthropogenic nitrogenous compounds. This catalytic response provides a selective advantage in terms of available nutrients while also serving to remove otherwise harmful xenobiotics. This review provides a critical assessment of the literature on bioconversion of organonitrogen compounds with a consideration of applications in bioremediation and commercial biotechnology. By examining the major nitro-organic compounds (amino acids, amines, nitriles, amides and nitroaromatics) in turn, the considerable repertoire of Rhodococcus spp. is established. The available published enzyme reaction data is coupled with genomic characterisation to provide a molecular basis for Rhodococcus enzyme activity with an assessment of the cellular properties that aid substrate accessibility and ensure stability. The metabolic gene clusters associated with the observed reaction pathways are identified and future directions in enzyme optimisation and metabolic engineering are assessed.Alanine dehydrogenase (RHA1_ro01495, ald1, ald2) and aspartate ammonia lyase (aspA1, aspA2, aspA3), with corresponding transaminases, provide alternative ping-pong (bi-bi) systems. Specificity and cellular catalytic efficiency is extended by a number of genes encoding asparagine dehydrogenase, arginine, cysteine, histidine and serine ammonia lyase ( Table 2). The production of

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Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540

Cited by 38 publications

References 31 publications

Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

ADP Competes with FAD Binding in Putrescine Oxidase

The repertoire of nitrogen assimilation in Rhodococcus: catalysis, pathways and relevance in biotechnology and bioremediation

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