Discovery of a eugenol oxidase from <i>Rhodococcus</i> sp. strain RHA1

Jin, Jianfeng; Mazon, Hortense; Heuvel, Robert H. H. van den; Janssen, Dick B.; Fraaije, Marco W.

doi:10.1111/j.1742-4658.2007.05767.x

Cited by 63 publications

(88 citation statements)

References 30 publications

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“…eugenol oxidase. For this enzyme, it was observed that incomplete FAD incorporation leads to the presence of apoenzyme rather than ADP-containing protein (19). This strongly suggests that incorporation of ADP in PuO Rh is likely to be the result of a competition between FAD and ADP binding, both being present in the cytoplasm.…”

Section: Discussionmentioning

confidence: 95%

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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Section: Discussionmentioning

confidence: 95%

ADP Competes with FAD Binding in Putrescine Oxidase

Hellemond

Mazon

Heck

et al. 2008

Journal of Biological Chemistry

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“…However, only a few GMC oxidoreductases can oxidize alcohols to the corresponding carboxylic acids (12,15). Although hydroxylations and amine oxidations have been described for other types of flavoprotein oxidases (16), thus far, no GMC flavoprotein oxidases with such activities have been identified (10).…”

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confidence: 99%

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

Dijkman

Fraaije

2014

Appl Environ Microbiol

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In the search for useful and renewable chemical building blocks, 5-hydroxymethylfurfural (HMF) has emerged as a very promising candidate, as it can be prepared from sugars. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a substitute for petroleum-based terephthalate in polymer production. On the basis of a recently identified bacterial degradation pathway for HMF, candidate genes responsible for selective HMF oxidation have been identified. Heterologous expression of a protein from Methylovorus sp. strain MP688 in Escherichia coli and subsequent enzyme characterization showed that the respective gene indeed encodes an efficient HMF oxidase (HMFO). HMFO is a flavin adenine dinucleotide-containing oxidase and belongs to the glucose-methanol-choline-type flavoprotein oxidase family. Intriguingly, the activity of HMFO is not restricted to HMF, as it is active with a wide range of aromatic primary alcohols and aldehydes. The enzyme was shown to be relatively thermostable and active over a broad pH range. This makes HMFO a promising oxidative biocatalyst that can be used for the production of FDCA from HMF, a reaction involving both alcohol and aldehyde oxidations.

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“…The eugenol catabolic pathway is substantiated by a previous characterization of eugenol oxidase (39). Nevertheless, the induction of these genes by ferulate is unexpected.…”

Section: Discussionmentioning

confidence: 60%

“…The first gene, eugO, encodes eugenol oxidase. This enzyme has been purified from RHA1 and oxidizes eugenol to coniferyl alcohol (39). Coniferyl alcohol is presumably oxidized to coniferyl aldehyde and then to ferulate (40).…”

Section: Growth Of Rha1 On P-hydroxycinnamatesmentioning

confidence: 99%

Characterization of p -Hydroxycinnamate Catabolism in a Soil Actinobacterium

et al. 2014

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p-Hydroxycinnamates, such as ferulate and p-coumarate, are components of plant cell walls and have a number of commercial applications. Rhodococcus jostii RHA1 (RHA1) catabolizes ferulate via vanillate and the ␤-ketoadipate pathway. Here, we used transcriptomics to identify genes in RHA1 that are upregulated during growth on ferulate versus benzoate. The upregulated genes included three transcriptional units predicted to encode the uptake and ␤-oxidative deacetylation of p-hydroxycinnamates: couHTL, couNOM, and couR. Neither ⌬couL mutants nor ⌬couO mutants grew on p-hydroxycinnamates, but they did grow on vanillate. Among several p-hydroxycinnamates, CouL catalyzed the thioesterification of p-coumarate and caffeate most efficiently (k cat /K m ‫؍‬ ϳ400 mM ؊1 s ؊1 ). p-Coumarate was also RHA1's preferred growth substrate, suggesting that CouL is a determinant of the pathway's specificity. CouL did not catalyze the activation of sinapate, in similarity to two p-coumaric acid:coenzyme A (CoA) ligases from plants, and contains the same bulged loop that helps determine substrate specificity in the plant homologues. The couO mutant accumulated 4-hydroxy-3-methoxyphenyl-␤-ketopropionate in the culture supernatant when incubated with ferulate, supporting ␤-oxidative deacetylation. This phenotype was not complemented with a D257N variant of CouO, consistent with the predicted role of Asp257 as a metal ligand in this amidohydrolase superfamily member. These data suggest that CouO functionally replaces the ␤-ketothiolase and acyl-CoA thioesterase that occur in canonical ␤-oxidative pathways. Finally, the transcriptomics data suggest the involvement of two distinct formaldehyde detoxification pathways in vanillate catabolism and identify a eugenol catabolic pathway. The results of this study augment our understanding of the bacterial catabolism of aromatics from renewable feedstocks.

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Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1

Cited by 63 publications

References 30 publications

ADP Competes with FAD Binding in Putrescine Oxidase

ADP Competes with FAD Binding in Putrescine Oxidase

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

Characterization of p -Hydroxycinnamate Catabolism in a Soil Actinobacterium

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