Characterization of hydroxy fatty acid dehydrogenase involved in polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009aby Michiki Takeuchi, Shigenobu Kishino, Si-Bum Park, Nahoko Kitamura, Jun Ogawa

Journal of Molecular Catalysis B: Enzymatic

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Year
2015
DOI
10.1016/j.molcatb.2015.03.020
Subject
Process Chemistry and Technology / Biochemistry / Bioengineering / Catalysis

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Text

Journal of Molecular Catalysis B: Enzymatic 117 (2015) 7–12

Contents lists available at ScienceDirect

Journal of Molecular Catalysis B: Enzymatic j ourna l ho me pa ge: www.elsev ier .com/ locate /molcatb

Characterization of hydroxy fatty acid dehydroge polyunsaturated fatty acid saturation metabolism plantar

Michiki T ah

Jun Ogaw a Division of Ap wakec b Laboratory of wa-oiw c Research Unit a r t i c l

Article history:

Received 2 Feb

Received in re

Accepted 31 M

Available online 6 April 2015

Keywords:

Lactic acid bacteria

Hydroxy fatty acid

Oxo fatty acid

Short-chain de ich is , was dent drogenation of hydroxy fatty acids. In the dehydrogenation reaction, fatty acids with an internal hydroxy group such as 10-hydroxy-cis-12-octadecenoic acid, 12-hydroxy-cis-9-octadecenoic acid, and 13-hydroxy-cis-9-octadecenoic acid served as better substrates than those with - or -hydroxy groups such as 3-hydroxyoctadecanoic acid or 2-hydroxyeicosanoic acid. The apparent Km value for 10-hydroxycis-12-octadecenoic acid (HYA) was estimated to be 38 M with a kcat of 7.6 × 10−3 s−1. The apparent Km 1. Introdu

Function pharmaceu functional f body fat co riosclerosis to have nov been repor juice acts as (PPAR) ag induced by

In our acid saturat [6], which ∗ Correspon

Graduate Scho 8502, Japan. T

E-mail add 1 These corr http://dx.doi.o 1381-1177/© hydrogenase/reductase value for 10-oxo-cis-12-octadecenoic acid (KetoA) was estimated to be 1.8 M with a kcat of 5.7 × 10−1 s−1.

In the hydrogenation reaction of KetoA, both (R)- and (S)-HYA were generated, indicating that the enzyme has low stereoselectivity. This is the first report of a dehydrogenase with a preference for fatty acids with an internal hydroxy group. © 2015 Elsevier B.V. All rights reserved. ction al lipids have attracted attention both nutritionally and tically. Conjugated linoleic acid (CLA) is a representative atty acid, which has beneficial effects such as decreasing ntent [1] and preventing tumorigenesis [2,3] and arte[4]. Oxo fatty acids as well as CLA have also been proven el physiological functions. For example, it has recently ted that 13-oxo-9,11-octadecadienoic acid in tomato a potent peroxisome proliferator activated receptor  onist and improves dyslipidemia and hepatic steatosis obesity [5]. previous study, we revealed polyunsaturated fatty ion metabolism in Lactobacillus plantarum AKU 1009a is a strain with a potential to produce CLA from ding author at: Kyoto University, Division of Applied Life Science, ol of Agriculture, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606el.: +81 75 753 6115; fax: +81 75 753 6113. ress: ogawa@kais.kyoto-u.ac.jp (J. Ogawa). esponding authors contributed equally to this work. linoleic acid [7–10]. The novel saturation metabolism consisted of four enzymes: CLA-HY (hydratase/dehydratase) [6,11,12], CLADH (dehydrogenase), CLA-DC (isomerase), and CLA-ER (enone reductase) [6,12]. This saturation metabolism included some oxo fatty acids, such as 10-oxo-cis-12-octadecenoic acid (KetoA), 10oxooctadecanoic acid, and 10-oxo-trans-11-octadecenoic acid, as intermediates. These oxo fatty acids are expected to have new physiological activities. CLA-DH generated these oxo fatty acids through dehydrogenation of the corresponding hydroxy fatty acids, e.g., dehydrogenation of HYA to KetoA.

In this study, we describe the enzymatic and physiochemical characteristics of CLA-DH, which is involved in the saturation metabolism and catalyzes the dehydrogenation of hydroxy fatty acids and the hydrogenation of oxo fatty acids. 2. Materials and methods 2.1. Chemicals

HYA, 10-hydroxyoctadecanoic acid, 10-hydroxy-trans-11octadecenoic acid, (S)-10-hydroxy-cis-12,cis-15-octadecadienoic acid, (S)-10-hydroxy-cis-6,cis-12-octadecadienoic acid, and rg/10.1016/j.molcatb.2015.03.020 2015 Elsevier B.V. All rights reserved.um AKU 1009a akeuchia, Shigenobu Kishinoa,b,1, Si-Bum Parkb, N aa,c,∗,1 plied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oi

Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Kitashiraka for Physiological Chemistry, Kyoto University, Kyoto, Japan e i n f o ruary 2015 vised form 23 March 2015 arch 2015 a b s t r a c t

Hydroxy fatty acid dehydrogenase, wh in Lactobacillus plantarum AKU 1009a preferentially catalyzed NADH-depennase involved in in Lactobacillus oko Kitamuraa, ho, Sakyo-ku, Kyoto 606-8502, Japan akecho, Sakyo-ku, Kyoto 606-8502, Japan involved in polyunsaturated fatty acid saturation metabolism cloned, expressed, purified, and characterized. The enzyme hydrogenation of oxo fatty acids over NAD+-dependent dehy8 M. Takeuchi et al. / Journal of Molecular Catalysis B: Enzymatic 117 (2015) 7–12 13-hydroxy-cis-9-octadecenoic acid were prepared as previously described [6,11,13,14]. Oxo fatty acids (KetoA, 10-oxooctadecanoic acid, 10-oxo-trans-11-octadecenoic acid, 10-oxo-cis-12,cis-15octadecadienoic acid, 10-oxo-cis-6,cis-12-octadecadienoic acid, 12-oxo-cisacid) were hydroxyoct acid, (S)-10 hydroxy-cis 13-hydroxy is oxidation purified fro column in hydroxy fa bovine ser

Louis, USA) commercia 2.2. Prepara

Escherich tured in 1 with simul thiogalacto 1.0 mM. Aft at 20 ◦C for vation, the standard bu (5 min, 4 t buffer conta potassium removed b supernatan extracts w 60 min and from this su (FPLC) syst buffer. The 200 prep-g equilibrated purified us

Superdex 2

Superose H with the sta −20 ◦C unti 2.3. Determ

In order

DH, the e mance gel(0.75 × 60 c eluted with a flow rate monitored determined teins. 2.4. Reactio

All oper standard re performed reaction m 0.1% (w/v) H substrate, 5

CLA-DH. One unit was defined as the amount of enzyme that catalyzes the conversion of 1 mol of HYA per minute. The reactions were performed under anaerobic conditions in a sealed chamber with an O2-absorbent (Anaeropack “Kenki,” Mitsubishi Gas Chem., Ltd