Nitrile Pathway Involving Acyl-CoA Synthetase

Hashimoto, Yoshiteru; Hosaka, Hideaki; Oinuma, Ken-Ichi; Goda, Masahiko; Higashibata, Hiroki; Kobayashi, Michihiko

doi:10.1074/jbc.m405686200

Cited by 34 publications

(28 citation statements)

References 58 publications

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“…For aromatic substrates, it is possible only to compare the enzyme activity of FadD from P. putida CA-3 to one other source, namely, a short-chain acyl-CoA synthetase from Pseudomonas chlororaphis B23 (9). P. putida CA-3 FadD exhibited a 13-fold-higher rate of reaction with 3-phenylpropionic acid than with propionic acid (Fig.…”

Section: Discussionmentioning

confidence: 99%

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

2009

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A fatty acyl coenzyme A synthetase (FadD) from Pseudomonas putida CA-3 is capable of activating a wide range of phenylalkanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher k cat and K m values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of the fadD gene from P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3⌬fadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3⌬fadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3⌬fadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of fadD in P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longermonomer units into the PHA polymer.Fatty acyl coenzyme A (CoA) synthetases (FACS; fatty acid: CoA ligases; EC 6.2.1.3) are ATP-, CoA-, and Mg 2ϩ -dependent enzymes that activate alkanoic acids to CoA esters for ␤ oxidation ( Fig. 1) (2, 17). FACS are widely distributed in both prokaryotic and eukaryotic organisms and exhibit a broad substrate specificity (34). FadD is a cytoplasmic membrane-associated FACS (7), with sizes ranging from 47 kDa to 62 kDa (2, 14). There is a lack of biochemical information on FadD with a preference for long-chain aromatic and aliphatic substrates. In the current study we purify and characterize for the first time a true long-chain FadD with activity toward both phenylalkanoic and alkanoic acids.It is known that bacteria such as Pseudomonas putida can accumulate the biological polyester polyhydroxyalkanoate (PHA) from aromatic as well as aliphatic alkanoic acids (5, 6, 42, 45). The presence of aromatic monomers in the PHA polymer suggests that a FadD with activity toward aromatic substrates is present in these PHA-accumulating strains. Garcia et al. knocked out an acyl-CoA synthetase in P. putida U with a high homology to long-chain fadD products from Escherichia coli and Pseudomonas fragi (6). Garcia et al. also showed that the mutant was not capable of growth or PHA accumulation with aromatic and aliphatic substrates having between 5 and 10 carbons in their acyl chain, indicating that it is a general and not a long-chain acyl-CoA ligase (6). In a follow-up study, Olivera et al. showed that the fadD mutants reverted to wild-t...

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Section: Discussionmentioning

confidence: 99%

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

2009

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“…The enzyme catalyzes the hydration of a nitrile to the corresponding amide followed by consecutive reactions: amide 3 acid 3 acyl-CoA by amidase (13) and acyl-CoA synthetase (14,15), respectively. Rhodococcus rhodochrous J1 produces high and low molecular mass NHases (H-NHase and L-NHase), which exhibit different physicochemical properties and substrate specificities (1,16).…”

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confidence: 99%

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

Zhou¹,

Hashimoto²,

Kobayashi

2009

Journal of Biological Chemistry

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The incorporation of cobalt into low molecular mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 has been found to depend on the ␣-subunit exchange between cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues) and its cobalt-containing mediator (holo-NhlAE containing Cys-SO 2 ؊ and Cys-SO ؊ metal ligands), this novel mode of post-translational maturation having been named self-subunit swapping, and NhlE having been recognized as a self-subunit swapping chaperone (Zhou, Z., Hashimoto, Y., Shiraki, K., and Kobayashi, M. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 14849 -14854). We discovered here that cobalt was inserted into both the cobaltfree NhlAE (apo-NhlAE) and the cobalt-free ␣-subunit (apo-␣-subunit) in an NhlE-dependent manner in the presence of cobalt and dithiothreitol in vitro. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis revealed that the non-oxidized cysteine residues in apo-NhlAE were posttranslationally oxidized after cobalt insertion. These findings suggested that NhlE has two activities, i.e. cobalt insertion and cysteine oxidation. NhlE not only functions as a self-subunit swapping chaperone but also a metallochaperone that includes a redox function. Cobalt insertion and cysteine oxidation occurred under both aerobic and anaerobic conditions when Co 3؉ was used as a cobalt donor, suggesting that the oxygen atoms in the oxidized cysteines were derived from water molecules but not from dissolved oxygen. Additionally, we isolated apo-NhlAE after the self-subunit swapping event and found that it was recycled for cobalt transfer into L-NHase.

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“…We reported as unpublished data 4 that A. pascens F164 showed isonitrile-degrading ability. This isonitrile-degrading enzyme coexists with NfdA in this strain.…”

Section: Discussionmentioning

confidence: 75%

“…Effects of pH and temperature on the activity of isonitrile hydratase. A, reaction was carried out for 10 min at 25°C in the following buffers (100 mM): citrate/potassium phosphate (OE), potassium phosphate (f), Tris-HCl (E), NH 4 Cl/NH 4 OH (Ⅺ), and KHCO 3 /KOH (‚). B, reactions were carried out for 10 min at various temperatures.…”

Section: Resultsmentioning

confidence: 99%

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Novel Isonitrile Hydratase Involved in Isonitrile Metabolism

Sato¹,

Hashimoto²,

Fukatsu

et al. 2010

Journal of Biological Chemistry

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We previously discovered N-substituted formamide deformylase (NfdA) in Arthrobacter pascens F164, which degrades N-substituted formamide (Fukatsu, H., Hashimoto, Y., Goda, M., Higashibata, H., and Kobayashi, M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13726 -13731). In this study, we found an enzyme involved in the first step of isonitrile metabolism, isonitrile hydratase, that hydrates isonitrile to the corresponding N-substituted formamide. First, we investigated the optimum culture conditions for the production of isonitrile hydratase. The highest enzyme activity was obtained when A. pascens F164 was cultured in a nutrient medium containing N-benzylformamide. This Arthrobacter isonitrile hydratase was purified, characterized, and compared with Pseudomonas putida N19-2 isonitrile hydratase (InhA), which is the sole one reported at present. Arthrobacter isonitrile hydratase was found to have a molecular mass of about 530 kDa and to consist of 12 identical subunits. The apparent K m value for cyclohexyl isocyanide was 0.95 ؎ 0.05 mM. A. pascens F164 grew and exhibited the isonitrile hydratase and N-substituted formamide deformylase activities when cultured in a medium containing an isonitrile as the sole carbon and nitrogen sources. However, both enzyme activities were not observed on culture in a medium containing glycerol and (NH 4 ) 2 SO 4 as the sole carbon and nitrogen sources, respectively. These findings suggested that the Arthrobacter enzyme is an inducible enzyme, possibly involved in assimilation and/or detoxification of isonitrile. Moreover, gene cloning of the Arthrobacter enzyme revealed no sequence similarity between this enzyme and InhA. Comparison of their properties and features demonstrated that the two enzymes are biochemically, immunologically, and structurally different from each other. Thus, we discovered a new isonitrile hydratase named InhB.We have extensively studied the biological metabolism of toxic compounds containing a CϵN moiety, such as nitriles (1-5) and isonitriles (6 -9). Isonitrile (more generally called isocyanide) containing an isocyano group (ϪNϵC) is an isomer of nitrile. Like nitrile, isonitrile is generally highly toxic and produced in nature, probably for self-defense, by various organisms, including bacteria, fungi, marine sponges, etc. (10, 11). For example, xanthocillin was first isolated as an isocyano metabolite from Penicillium notatum, and this isonitrile exhibits a wide antibiotic activity spectrum (12). There have been many other reports about such naturally occurring isocyanides, but information is quite limited on the metabolism of isonitrile. Some metabolic isonitrile intermediates have been elucidated through incorporation experiments, and we initially reported two enzymes involved in isonitrile metabolism. First, we discovered isonitrile hydratase in Pseudomonas putida N19-2, which catalyzes the hydration of an isonitrile to the corresponding N-substituted formamide (R-NH-CHO) (6, 7). It has been approved as a new enzyme by the Nomenclature Committee of t...

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Nitrile Pathway Involving Acyl-CoA Synthetase

Cited by 34 publications

References 58 publications

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

Novel Isonitrile Hydratase Involved in Isonitrile Metabolism

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