A new biosynthetic pathway of porphyrins from isopropanol

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 77, NO. 6, 626-629. 1994

A New Biosynthetic Pathway of Porphyrins from Isopropanol MASAHIRO KAJIWARA, 1. MINORU MIZUTANI, l REIKO MATSUDA, 1 KEN-ICHIRO HARA, l AND ICHIRO KOJIMA 2

Department of Medicinal Chemistry, Meiji College of Pharmacy, 1-22-1 Yato-cho, Tanashi-shi, Tokyo 1881 and Central Technical Research Laboratory, Nippon Oil Company, Ltd., 8, Chidori-cho, Naka-ku, Yokohama 231,2 Japan Received 31 January 1994/Accepted 28 February 1994

A new biosynthetic pathway, which can produce both vitamin Bn and large amounts of porphyrins from isopropanol, was identified in Arthrobacter hyalinus using carbon-13 stable isotope tracer techniques and carbon13 nuclear magnetic resonance (t3C-NMR) spectroscopy. Studies on the incorporation of [2-13C]isopropanol, [I- or 2-13C]sodium acetate, L-[l-13C]glutamate, and [1-, 2-, 3-, 4-, 5-13C]5-aminolevulinic acid into uroporphyrinogen III showed that isopropanoi was metabolized into uroporphyrinogen III through acetyl CoA and that 5-aminolevulinic acid was produced from L-glutamic acid and not via Shemin's pathway.

A bacterium, Arthrobacter hyalinus, which produces 2 mg/l vitamin B~2, was isolated and a mutant which produces 440 mg/l coproporphyrin III, 170 mg/l uroporphyrin III, 25 mg/l uroporphyrin I and a small amount of penta-, hexa-, and hepta-carboxylic porphyrins from isopropanol (IPA) as a sole carbon source has been constructed (3, 4). Ueyama et al. isolated an Arthrobacter sp. which produces acetone from IPA (5, 6). Sato et al. isolated a Mycobacterium sp. which produces a-ketobutyric acid from IPA (7). Yeasts such as Candida, Hansenula, Pichia, Torulopsis, and Kloeckera have been reported to produce acetone from IPA although they could not assimilate IPA (8-10). However, there are no reports indicating that IPA is a good source for the production of vitamin B~2 and porphyrins. In this report, we describe a new pathway for the production of uroporphyrin III and vitamin B12, whose precursor is a reduced form of uroporphyrin III, from IPA through acetic acid, L-glutamic acid and 5-aminolevulinic acid (ALA). A description of ALA produced via the C-5 pathway, in which ALA is produced from L-glutamic acid, is also presented.

glutamate, or 50mg of [1-, 2-, 3-, 4-, 5-13C]ALA was shaken at 30°C for 7-10d after being inoculated with

A. hyalinus. Isolation of uroporphyrinogen III The culture broth was centrifuged (10,000 z g, 10 min) to remove the bacterial cells and DEAE Sephadex A-25 gel was added to the supernatant to adsorb uroporphyrin III which formed from uroporphyrinogen III during the culture. The gel was freeze-dried and suspended in dry methanol containing 5% sulfuric acid. The uroporphyrin III octamethylester which formed was purified by silica gel chromatography and recrystalization in chloroform and methanol (4). 1H-NMR and t3C-NMR 1H-NMR spectra and 13CNMR spectra were taken on a JEOL GS-400 spectrometer (400 and 100 MHz) (Tokyo). Chemical shifts are given down field from tetramethylsilane (TMS) in the case of ~H-NMR, and from chloroform-dl (=77.0 ppm) as an internal standard in the case of ~3C-NMR. RESULTS AND DISCUSSION Incorporation of [2-13C]IPA A. hyalinus was cultured in medium containing [2J3C]IPA [(la) in Fig. 1], and uroporphyrin III octamethylester was isolated from the supernatant and examined by 13C-NMR. ~3C-Enriched signals were observed at 171.9 ppm (COOCH3 × 4, s; acetyl carboxylic carbons), 173.5 ppm (COOCH3 × 4, s; propyl carboxylic carbons) and 98.1 ppm (aromatic CH × 4, m; meso carbons of porphyrin ring) (Fig. 2). The two former signals showed that the 2-C (o) of IPA was incorporated into 8 carboxyl groups of uroporphyrinogen III through 1-C of a-ketoglutaric acid, while the last signal showed that the 4-C (a) of succinate was incorporated into 8 carboxyl groups of uroporphyrinogen III from the second cycle of the TCA cycle since 2 types of labeled succinate such as [IJ3C] and [4-~3C]succinate are equally formed in the first cycle. Incorporation of [1-13Clsodium acetate 13C-NMR analyses of uroporphyrin III octamethylester isolated from the culture broth using the medium supplemented with [1-13C]sodium acetate showed that enriched signals were also seen at 171.9 ppm (COOCH3 × 4, s; acetyl carboxylic carbons), 173.5 ppm (COOCH3 × 4, s; propyl carboxylic carbons) and 98.1ppm (aromatic C - H × 4 , m;

MATERIALS A N D METHODS 13C-labeled compounds [2-13C]IPA was prepared by reducing [2-13C]acetone using LiA1H4 at a yield of 92%. N-Acetyl DL-[1-~3C]glutamic acid was synthesized from [~3C]potassium cyanide and was hydrolyzed by acylase to form L-[l-~3C]glutamic acid (11). [1-, 2-, 3-, 4-, 5-~3C]ALA were prepared from t3C-labeled glycine, Meldrum's acid, or bromoacetate (12). Other stable isotopelabeled compounds such as [1- or 2-13C] sodium acetate and [13C]potassium cyanide were purchased from Cambridge Isotope Laboratories (Woburn, USA), and Merck & Corporation (Quebec, Canada), respectively. Feeding experiments of [2-13CllPA, [I- or 213C]sodium acetate, L-[l-13C]glutamate, and [!-, 2-, 3-, 4-, 5-13C]5-aminolevulinic acid in A. hyalinus A 500 ml conical flask containing 200 ml of porphyrin production medium (4) supplemented with 2 ml of [2-13C]IPA, 1.0 g of [1- or 2-~3C]sodium acetate, 150mg of L-[1-13C] * Corresponding author. Biosynthesis of Corrinoids and Porphyrinoids (VIII-1, 2).

626

VOL. 77, 1994

BIOSYNTHETIC PATHWAY OF PORPHYRINS

627

pro .S

• COOH C H 3 " ! ~

HO-C-COOH CH2 @

pro -R

• COOH

(3a)

I O=C

•

CH3-CH'CHa OH

CH2 • COOH

(la)

Oxaloecetlc acid

• : First cycle • : Second cycle

~ ,

• COOH CH2

Citric acid

CH~ TCA

•

-

C=O COOH

COOH I

CH~ CH~

_)

• COOH $ucclnl¢ acid

CH~

.COOH CIH2 .

H-C-NH2 • COOH

ct-Katoglutarlc acid

•

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L-Glutamlc acid (4)

_

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t'OOH

" 1 ~ N")i/ NH2 H

NH2 AL& (S)

~...NH. ,N~ HOOC'~,II-t~.,y/-~COOH

PBG (6)

•

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(7)

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=

• C=O

CH2 NH2

I J

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Gly¢lna

I

HOOC

.

ALA

Shemin route

FIG. 1. Hypothetical biosynthetic pathway of uroporphyrinogen III in A. hyalinus. meso carbons o f porphyrin ring). These incorporations were consistent with the [2-13C]IPA incorporation. This fact indicates that I P A was metabolized through acetate into uroporphyrinogen III. Incorporation of t-[l-z3C]glutamic acid A L A (5 in

Fig. 1) is synthesized from glycine and succinyl C o A (Shemin's pathway) in mammals (13-15), Rhodopseudomonas spheroids (16) and a yeast (17). In contrast, it is synthesized from L-glutamic acid (C-5 pathway, in Fig. 1) in plants (18-22), E. coli (23) and Salmonella

•

CO (A)

CH3-C,H -CH3 OH [2-13C11PA (la)

1713 ppm

/

Co (I,) 173.5 ppm

H3CO2.c ~

esterification

H3°°4

"o2o,3 oo20

meso (A)

• ~"C02CH3

co c.

Uroporphyrin III octamethylester (8) .

1

Natural

lljj PPM 1 ' I I I I L I I ' ' 1 140 ~ 100 BO 60 40 20 0 FIG. 2. Comparison of the ~3C-NMR spectrum (100 MHz: solvent CDCI~) of [2J3C]isopropanol (la)-incorporating uroporphyrin III octamethylester (A) with the natural abundance spectrum (B). I

IBO

~

I

~BO

628

KAJIWARA ET AL.

J. FERUmNT.BIO~NO.,

H3CO2(~ HO=~ Arthrobacter hyalinus ~NH N=~ x.-CO2CH~ ~O~H .sterifieation ~ ' - ~ " ~ ' - x L-[1-t3C]Glutamic acid (4)

H~(3020 C02CH3 Uropotphyrin rff octm'ae~ylestcr(Be)

143.9ppm

meso

l ~.1 ppm

(A)

Natural

~ (B)

C13

J1

~ ilI

~H 200

t50

t00

50

0

FIG. 3. Comparison of the '3C-NMR spectrum (100MHz: solvent CDCI3) of ~.-[1-'sC]glutamatc (4)-incorporating uroporphyrin III octamethylester (A) with the natural abundance spectrum (B).

typhimurium (24). '3C-NMR analyses of uroporphyrin III octamethylester isolated from the culture broth using the medium supplemented with L-[1-13C] glutamate (4 in Fig. 1) showed that enriched signals were observed at 98.1ppm (m; meso carbons) and 143.9ppm (pyrrolic ring a carbons) (Fig. 3). These incorporations were consistent with the [5-~3C] (,,) ALA incorporation, that indicating that L-glutamic acid was metabolized into uroporphyrinogen III through ALA via the C-5 pathway. If L-glutamic acid was not utilized and succinate and glycine were utilized for porphyrin production, the meso carbons of the porphyrin ring would not have been

•

o

•

H3CO,C

~

~

COCH3 • =~"/cO2CH3

~

"CO2CH3

labeled although the carboxylic carbons of porphyrin would have been labeled through [1-13C] and [4-13C]ALA (Fig. 1). 13C-NMR analyses of uroporphyrin III octamethylester isolated from the culture broth using the medium supplemented with [2-1aC]sodium acetate showed that enriched signals were also observed at 32.6 ppm (CHECO, s; a methylene carbons in acetyl groups), 37.1ppm (CHECHECOO , S; O~ methylene carbons in propyl groups), 21.9ppm (CH2CH2COO , s; ~ methylene carbons in propyl groups) and 133.1 and 144.1ppm (pyrrolic ring ~ carbons). Furthermore, 13C-NMR analyses of uroporphyrin III octamethylester isolated from the culture broth using the medium supplemented with [1-, 2-, 3-, 4-, and 5-13C] ALA (Sa-e in Fig, 4) indicated that they were incorporated into uroporphyrinogen III after conversion to the uroporphyrin III octamethylester (8a-e in Fig. 4). These results are consistent with the hypothesis that IPA is successively transformed into acetyl-CoA, TCA cycle products (25), z-glutamate, ALA, PBG, and uroporphyrinogen III, and rule out the involvement of Shemin's pathway to any significant degree in A. hyalinus, as summarized in Fig. 1. ACKNOWLEDGMENT

ALA

(5a-e)

III octamethylester (8a-e)

Uroporphyrin

FIG. 4. The incorporation of ~3C-labeled ALA (Sa-5e) into uroporphyrin III octamethylester (8a-8e). Symbols: o, (Sa-*Sa); *, (Sb--*8b); *, (5c-~8c); &, (Sd~,8d); m, (Se-~8e).

We are grateful to Prof. T. Imanaka, Department of Biotechnology, Osaka University, for his critical reading of this paper and valuable advice.

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