human carbonyl and aldose reductases: new catalytic functions in tetrahydrobiopterin biosynthesis

Vol. 175, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 29, 1991 Pages 738-744

HUMAN CARBONYL AND ALDOSE REDUCTASES: NEW CATALYTIC FUNCTIONS IN TETRAHYDROBIOPTERIN BIOSYNTHESIS

Y. S. Park, C. W. Heizmann, B. Wermuth’, R. A. Levine*, P. Steinerstauch, J. Guzman, and N. Blau

Department of Pediatrics, Division of Clinical Chemistry, University of ZSrich, CH-8032 Ziirich, Switzerland

‘Department of Clinical Chemistry, University of Bern, CH-3010 Bern, Switzerland

*Laboratory of Molecular Neurobiology, Lafayette Clinic, and Department of Psychiatry, Wayne State University, Detroit

Received February 8, 1991

SUMMARY: New catalytic functions of human carbonyl- and aldose reductase in tetrahydrobiopterin biosynthesis are proposed. 6-Pyruvoyl tetrahydropterin, an intermediate in the biosynthesis of tetrahydrobiopterin, was converted to 6-lactoyl tetrahydropterin and 1 ‘-hydroxyQ’- oxopropyl tetrahydropterin by carbonyl reductase under anaerobic condition. 1 ‘-HydroxyQ’- oxopropyl tetrahydropterin was subsequently metabolized to tetrahydrobiopterin by aldose reductase. Based on these results alternative pathways for the synthesis of tetrahydrobiopterin in patients with genetic defects of sepiapterin reductase are suggested. 0 1991 Academic

Press, Inc.

INTRODUCTION: The biosynthesis of tetrahydrobiopterin (BH,) involves several enzymatic steps.

This includes the reduction of 6-pyruvoyl tetrahydropterin (PPH,) to l’-hydroxy-2’-oxopropyl

tetrahydropterin (2’-0x0-PH,) by sepiapterin reductase and the reduction of PPH, to 6-lactoyl

tetrahydropterin (LPH,) by aldose reductase (AR, formerly named PPH, reductase) (1-9). Both

tetrahydropterins are further reduced to BH, by sepiapterin reductase. BH, is the cofactor for the

monooxygenases which hydroxylate phenylalanine, tyrosine and tryptophan to the precursors of

the catecholamine and indoleamine neurotransmitters (10). A deficiency of BH, is known to

cause hyperphenylalaninemia leading to abnormal development of newborns. Three enzyme

defects have been identified that lead to BH, deficiency (11,12); these are a deficiency in GTP

cyclohydralase I, PPH, synthase, and dihydropteridine reductase. Thus far, a sepiapterin

reductase deficiency in newborns has not been reported, which could be due to : (a) sepiapterin

reductase deficiency does not allow a fetus to survive, or, (b) sepiapterin reductase deficiency is

ABBREVIATIONS: BH,, tetrahydrobiopterin; NH,TP, 7,Bdihydroneopterin triphosphate; PPH,, 6- pyruvoyl tetrahydropterin; LPH,, 6-lactoyl tetrahydropterin; 2’-0x0-PH,, 1 ‘-hydroxy-2’-oxopropyl tetrahydropterin; H, pterins, tetrahydropterins; NAS, N-acetyl serotonin; PPH, synthase, 6-pyruvoyl tetrahydropterin synthase; AR, aldose reductase; CR, carbonyl reductase; SR, sepiapterin reductase.

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in arty form reserved. 738


compensated by other reductases with similar properties. Our results strongly support the second

hypothesis suggesting new catalytic functions of carbonyl- and aldose reductases in BH,

biosynthesis.

MATERIALS AND METHODS

BH, and sepiapterin were purchased from Dr. B. Schircks Laboratories (Jona, Switzerland). 7,8-Dihydroneopterin triphosphate (NH,TP) was prepared using E. co/i GTP cyclohydrolase I immobilized to affinity support (13). LPH, and 2’-0x0-PH, standards were prepared following the methods described (14, 15). Catalase (beef liver), glucose oxidase (Aspergillus niger), and NADPH were from Boehringer Mannheim, FRG.

Preparation of enzymes: Aldose- and carbonyl reductase were purified as described elsewhere (16,17). Sepiapterin reductase was partially purified from human brain according to the method described previously (18). For each enzyme preparation the absence of the other two reductases was confirmed by immunoblot analysis using antibodies prepared against aldose reductase (19) carbonyl reductase (16), and sepiapterin reductase (20). PPH, synthase was purified from salmon liver (21).

Analysis of tetrahydropterins by HPLC with electrochemical detection and the preparation of the substrate, PPH,, were performed as described previously (16) with modifications: The standard reaction mixture was 0.1 M potassium phosphate buffer, pH 7.0, 10 mM dithioerythritol, 8 mM MgCI,, 14.5 PM NH,TP, 2 mU PPH, synthase, and 2 mM NADPH. Anaerobic conditions were generated by adding 10 Mm glucose, glucose oxidase (150 units/ml), and catalase (>5000 units/ml) to the reaction mixture. PPH,, generated in situ, was used without further purification.

2’-0x0-PH, and LPH, were prepared by addition of sepiapterin reductase (0.45 mU/ml reaction mixture), and aldose reductase (0.126 mu/ml reaction mixture), to the reaction mixture described above. The mixtures were incubated for 90 min at 37.5% and filtered through an Amicon centrifuge concentrator (Centricon-3) to remove the enzymes. All the preparations were made fresh just before use.

Enzyme assays: Enzyme activities using sepiapterin as a substrate were assayed using HPLC with fluorescence detection (22). Enzyme activities using PPH, as a substrate were measured as follows: The reaction mixture of PPH, (see above) and enzyme in a total volume of 100 ul was prepared in autosampler vials, flushed with nitrogen, sealed and incubated at 37.5%. Reactions were terminated by chilling in ice cold water and samples were immediately injected to HPLC. BH, was used as an external standard to quantify LPH, and 2’-0x0-PH,. Enzyme activities with LPH, and 2’-0x0-PH, as substrates were assayed in reaction mixtures supplemented with NADPH, dithioerythritol, glucose, glucose oxidase, and catalase to the final concentrations as described in the preparation of PPH,. Enzyme units are defined as umol product generated per minute.

RESULTS AND DISCUSSION

In an attempt to purify sepiapterin reductase from human brain another enzyme was co-

purified which also used sepiapterin as a substrate but with biochemical properties different from

sepiapterin reductase. Gel permeation chromatography of a highly purified preparation of this

enzyme yielded a single peak of enzyme activity with a molecular weight of 32’000 (Fig. 1). This

enzyme was identified as human carbonyl reductase by several criteria including Western blot

analysis using antibodies against the three major low molecular weight human aldo-keto

reductases distinguished by different pl’s (19). Table 1 lists the specific activity of this enzyme

739


02 0,25

A BC D 3

- 0,2 - . 0,15 -

i I llrl

2 1234

1

g 0,l

- 0,15 j$

3 E

b

ii

- 0,l s .c-

n .? 5 0.05 - ij m

2

- 0.05 4

0 0 0 20 40

Fraction Number 80 100

Flaure 1. Gel permeation chromatography of carbonyl reductase. The enzyme was prepared from human brain extracts (350 g of cortex) by ammonium sulfate fractionation (40-55 %), and consecutive chromatography on Matrex-Red A (Amicon), DEAE-Sephadex A50 (Pharmacia), Affigel-Blue (Bio-rad), and Serotonin agarose (Sigma). The final sample (102 mu) was applied to a Sephadex G-150SF (Pharmacia) column (2.6x94 cm) and eluted with 20 mM potassium phosphate, 200 mM KCI, pH 6.8. Fractions (5.8 ml) were collected every hour and assayed for protein and activity to sepiapterin as described in “Materials and Methods”. Fractions with activity were pooled, concentrated by using ultrafiltration (Amicon), and stored at -20°C. The molecular weight markers; A: bovin serum albumin (MW 67’000), B: ovalbumin (MW 43’000), C: chymotrypsinogen(MW25’OOO),D: ribonuclease(MW 13’700). Insert: lmmunoblot of fraction with sepiapterin reductase activity (1) and multiple forms of carbonyl reductase (3-4). Proteins (appr.0.2 pg each) were separated by SDS-PAGE and detected using antibodies against human carbonyl reductase.

preparation compared with those of carbonyl reductase from human brain. The rates of reduction

of sepiapterin by sepiapterin- and carbonyl reductase are comparable. However, when menadione

was used as a substrate they were about five hundred-fold lower for both enzymes. Nevertheless,

when human brain extract was incubated with antibodies against carbonyl reductase, sepiapterin

Table 1. Actlvltles of carbonyl and aldose reductase uslng sepiapterin and PPH, as substrates

production of biopterin production of tetrahydropterins from sepiapterin from PPH,

nmol/mg proteinlmin LPH, 2’-0x0-PH, nmol/mg protein/min

CR’ CR IEF 7.5’ CR IEF 8.0’ CR IEF 8.5’ Aldose reductase (42 mU/mg protein)3

16.10 1.14 0.14 9.10 0.34 0.22 7.50 0.52 0.12

15.00 1.72 0.23 0.77 42.00

’ Enzyme purified from human brain as decribed in Fig.1. ’ Multiple forms of human brain carbonyl reductase, CR (17); IEF (isoelectic point). 3 Purified from human liver (16).

740


reduction was inhibited more than 30% (data not shown), indicating that carbonyl reductase

significantly contributes to the total sepiapterin reductase activity of human brain.

These findings encouraged us to examine a possible catalytic function of carbonyl reductases

in the conversion of PPH,, the natural substrate of mammalian sepiapterin reductase. In the

presence of the enzymatic oxygen-depleting system used to measure the synthesis of

tetrahydropterins from PPH,, two major peaks of enzymatic products were found by HPLC. These

two peaks were identified as LPH, and 2’-0x0-PH,, by comparison with reference compounds. The

identity of the two compounds was further verified by adding sepiapterin reductase to the assay

mixture. In keeping with the known substrate specificity of this enzyme, both products were

completely converted to BH, (Fig 2A). The results of PPH, reduction by carbonyl- and aldose

reductase are summarized in Table 1. In agreement with our earlier report (9), LPH, was the only

product of aldose reductase-mediated PPH, reduction. In contrast, carbonyl reductase catalyzed

the reduction of both the l’- and 2’-0x0 group of PPH, although LPH, was the prevailing product.

Consistently, small amounts of BH, (which increased with the incubation time) were detectable

in the assay mixture. Similar BH, production was also noticed with aldose reductase as catalyst,

as well as in the control experiments (absence of the enzyme). Moreover, when carbonyl

reductase (0.03 mu) was incubated with either LPH, or 2’-0x0 PPH,, no BH, was produced.

The carbonyl reductase-catalyzed reduction of PPH, was stimulated by DTE with a markedly

more pronounced effect on the synthesis of 2’-0x0 PPH,. At concentrations of 1 and 5 mM DTE

the production of 2’-0x0-PH, was increased by 186%, and 215%, whereas the LPH, level was

increased by 125%, and 120%, respectively. The effect of DTE probably reflects the known

sensitivity of carbonyl reductase to sulfhydryl reagents (17). This does not explain, however, the

differential stimulation of LPH, and 2’-0x0-PH, production. N-acetyl serotonin, an inhibitor of

sepiapterin reductase, only slightly inhibited carbonyl reductase. At 5 mM, a concentration which

completely inhibits rat sepiapterin reductase, carbonyl reductase was still 80% active.

Aldose reductase, in addition to converting PPH, to LPH,, also catalyzed the reduction of

2’-0x0-PH, to BH,, as demonstrated in Fig. 28. In the presence of catbonyl reductase, an

alternative pathway converting PPH, to BH, in the absence of sepiapterin reductase could be

functional in vivo. The plausibility of this pathway in vitro is documented in Fig. 2C. 2’-0x0-PH.,

synthesized by carbonyl reductase was selectively and completely converted to BH, by aldose

reductase. The concomitant decrease of LPH,, on the other hand, reflects the instability of this

compound rather than its enzymatic reduction as verified by a control incubation in the absence

of aldose reductase.

The biological significance of the alternative pathway in vivo is difficult to estimate. On the one

hand, human brain contains large amounts of aldose (25) and carbonyl (17) reductases, which

are both found at similar locations throughout the brain. On the other hand, only a small portion

of PPH, is converted to 2’-0x0-PH, by carbonyl reductase and the rate of reduction is much lower

than that of sepiapterin reductase. Therefore, in BH, deficiency caused by a putative genetic

741

Vol. 175, No. 3, 1991 BIOCHEMICAL AND BIOPI-IYSICAL RESEARCH COMMUNICATIONS

.Jd

0 1

20 40

10 mln 6 I

Omln

Retention time ( min )

Fiaure 2. Reduction of tetrahydropterins by carbonyl and aldose reductase. PPH, was incubated with either carbonyl reductase (0.39 mu/ml reaction mixture) (A,C) or low concentrations of sepiapterin reductase (B) as indicated in “Materials and Methods”. After ultrafiltration (Amicon) sepiapterin reductase (0.45 mu) was added to filtrate A and aldose reductase (0.07 mu) to filtrate B and C. Controls without addition of the second enzyme were routinely included. At times indicated (20 min for controls) aliquots were subjected for HPLC on (4.6 x 250 mm) ODSl Spherisorb column (Stagroma, Switzerland) using 6.6 mM sodium hydrogenphosphate, 13.3 mM citric acid, 0.06 mM EDTA. 1.4 mM octanesulfonic acid, 0.16 mM DTE and 5% methanol as solvent (1 ml/min) and electrochemical detection at a sensitivity of WA.

defect of sepiapterin reductase two cases must be distinguished. In the case of a sepiapterin

reductase deficiency with residual activity the following pathways would be possible:

AR and/or CR (SW PPH, _________________________________ > LPH, ------------------------> BH,

AND/OR

(SW AR

PPH, ---------------------------------> 2’-0x0-PH, __________________ > BH,

742


Residual sepiapterin reductase which preferentially catalyzes the reduction of the I’-0x0

group, could produce enough 2’-0x0-PH, to serve as a substrate of aldose reductase and also

convert LPH, produced from PPH, by the action of aldose reductase and carbonyl reductase to

BH,. In this case, the decreased activity of sepiapterin reductase might be fully compensated by

aldose- and carbonyl reductase.

In the case of a complete deficiency of sepiapterin reductase, BH, could only be synthesized

by the concerted action of aldose- and carbonyl reductases or by any other hitherto unidentified

enzyme catalyzing the reduction of the various tetrahydropterins:

CR AR

PPH, _________________________________ > 2’-0x0-PH, __________________ > BH,

Further investigations using antibodies against human sepiapterin reductase will be initiated

in order to clarify the significance of the proposed alternative pathway and the potential for a

genetic deficiency of sepiapterin reductase.

ACKNOWLEDGMENTS: We thank Mr. W. Leimbacher and Mr. F. Neuheiser for providing

excellent technical assistance. Y. S. Park is a Visiting Professor from The Department of

Microbiology, lnje University, Kimhae 621-749, Korea, supported in part by a fellowship from the

Korea Science and Engineering Foundation. This work was supported by the Swiss National

Science Foundation (Grant No. 31-26609.89 and 31-28797.90).

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human carbonyl and aldose reductases: new catalytic functions in tetrahydrobiopterin biosynthesis

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