Diversity and versatility of microbial hydantoin-transforming enzymes

JOURNAL OF MOLECULAR CATALYSIS 6: ENZYMATIC Journal of Molecular

Catalysis 8: Enzymatic

2 ( 1997) I63- 176

Review

Diversity and versatility of microbial hydantoin-transforming enzymes Jun Ogawa, Sakayu Shimizu Department

of Agricultural

Chemistry,

Facula

of A,qriculture.

Kxoto

x Unil~ersir~, Kyto

606. Japan

Received 8 July 1996; revised 14 August 1996; accepted 28 August 1996

Abstract Microbial hydantoin transformation has been applied to produce optically active amino acids. The transformation involves ring-opening hydrolysis of cyclic ureides and successive hydrolysis of N-carbamoyl amino acids. The enzymes catalyzing these two hydrolytic reactions were purified from various microorganisms and characterized. In the N-carbamoyl amino acid hydrolysis, three enzymes, N-carbamoyl-D-amino acid amidohydrolase, N-carbamoyl-t-amino acid amidohydrolase and P-ureidopropionase, are involved. The former two enzymes only hydrolyze N-carbamoyl-o-amino acids D- or L-stereospecifically, respectively. The last one acts upon N-carbamoyl-cu-, -p- and -y-amino acids, and shows L-stereospecificity to N-carbamoyl-o-amino acids. A variety of enzymes are also involved in cyclic ureide hydrolysis. D-Hydantoinase hydrolyzes Smonosubstituted hydantoins D-stereospecifically and preferably hydrolyzes dihydropyrimidines. Imidase, which acts well upon cyclic imides, also hydrolyzes dihydropyrimidines. N-Methylhydantoin amidohydrolase hydrolyzes Smonosubstituted hydantoins L-stereospecifically with concomitant hydrolysis of ATP to ADP. Dihydroorotase L-stereospecifically. The strict stereospecificities of these hydrolyzes six-membered cyclic ureide, dihydroorotate, hydantoin-transforming enzymes contribute to produce optically active compounds. Keywords:

Hydantoin

transformation;

Ureides; Carbamoyl

amino acid: Ring-opening

1. Introduction Hydantoins are commonly used in organic synthesis. The most important members of this family are the Smonosubstituted hydantoins, which are used as precursors for the chemical synthesis of rx-a-amino acids [l]. Based upon the investigations of Dudley et al. [2,3] into the metabolism of N-substituted DL-Sphenylhydantoins, which were postulated

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1381-l 17/97/$17.00 Copyright S 138 l-l 177(96)00020-3

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753 6115, Fax: (+81-

hydrolysis

to be o-stereoselective, Cecere et al. [4] found in 1975 that dihydropyrimidinase from calf liver could be used to produce several N-carbamoylo-amino acids from the corresponding DL-5 monosubstituted hydantoins. In 1978, Yamada and co-workers showed that microbial cells are good catalysts for this reaction [5-91. Thereafter, several bacteria which could hydrolyze N-carbamoyl-D-amino acids to o-amino acids were found [lo]. With these microorganisms, enzymatic production of o-amino acids from ix-5-monosubstituted hydantoins were established.

0 1997 Elsevier Science B.V. All rtghts reserved

164

J. Ogawa, S. Shimizu/Jounal

of Molecular Catalysis B: Enzymatic 2 (1997) 163-176

Similar reactions transform DL-5-monosubstituted hydantoins to L-amino acids. Yokozeki et al. [ll] analyzed the reaction mechanism of L-tryptophan production from DL-5-indolylmethylhydantoin by Flauobacterium sp., and found that non-stereospecific hydrolysis of hydantoin derivative and the L-selective hydrolysis of racemic N-carbamoyl amino acid were involved in this transformation. Yamashiro et al. [ 121found the L-selective hydrolysis of a hydantoin derivative in L-valine production from DLSisopropylhydantoin by Bacillus sp. and that ATP was absolutely required for this amidohydrolytic reaction. Despite the practical importance of these hydantoin-transforming reactions, the enzymes involved in these reactions have never been studied in detail except for the D-5-monosubstituted hydantoin-hydrolyzing enzymes, which are called D-hydantoinases. This review describes our efforts to reveal in detail the characters of the enzymes involved in D- or L-amino acids production from DL-5-monosubstituted hydantoins. This information is valuable for elucidating the physiological roles of these useful enzymes, and for broadening the applications of these enzymes.

2. Optically pure a-amino acid production by microbial hydantoin-transforming enzymes D-p-Hydroxyphenylglycine and its derivatives are important as side-chain precursors for semisynthetic penicillins and cepharosporines. Yamada and co-workers found that these amino acids can be efficiently prepared from the corresponding 5-monosubstituted hydantoins using microbial enzyme, D-hydantoinase [5]. Interestingly, the enzyme attacked a variety of aliphatic and aromatic D-5-monosubstituted hydantoins, yielding the corresponding D-form of Ncarbamoyl-cr-amino acids. Thus the enzyme can be used for the preparation of various D-amino acids.

IAmkloslkyhtlon

(D-Hydantoinaee

CCOH

HqNHCONH

*

Chemical decarbemoylatlon or N-Carbamoyl-D-amino amldohydrolase

acid H

a COOH Q

Fig. 1. pp-Hydroxyphenylglycine

H

production

NH2

process.

Initially, the synthetic process for D-p-hydroxyphenylglycine involves two chemical steps and one enzymatic step [13] (Fig. 1). The subis strate, DL-5-c p-hydroxyphenyl)hydantoin, synthesized through an efficient chemical method involving the amidoalkylation reaction of phenol with glyoxylic acid and urea under D-5-( pconditions. Then, acidic hydroxyphenyl)hydantoin is hydrolyzed enzymatically to N-carbamoyl-D-p-hydroxyphenylglycine. Under the conditions used for the enzymatic hydrolysis of hydantoin at pH 8 to 10, the remaining of the L-isomer 5-t phydroxyphenyl)hydantoin is racemized by base catalysis. Therefore, the racemic hydantoins can be converted quantitatively into N-carbamoyl-Dp-hydroxyphenylglycine through this step. Decarbamoylation to D-p-hydroxyphenylglycine was performed by treating the N-carbamoyl-Damino acid with equimolar nitrite under acidic conditions [14]. But now, this step can also be carried out enzymatically. Recently, a novel enzyme, which stereospecifically hydrolyzes N-carbamoyl-D-amino acids, was found in several bacteria [ 15,161. Therefore, a sequence of

J. Ogawa,

S. Shimizu/Joumal

of Molecular

Catalysis

B: Enzymatic

3. Evaluation of hydantoin- and pyrimidinetransforming activities in bacteria DL-5-Monosubstituted hydantoins are converted to o-amino acids via N-carbamoyl-Damino acids by some bacteria through the pathway shown in Fig. 2A [19-211. Takahashi et al. [6] revealed that, in Pseudomonas putida ( = P. striatu) IF0 12996, D-hydantoinase is identical

NH,

0 R

4

HN4;”

R W)

F

=

0

“20

NH \

COO" 4o%

R-_?:

r

COOH “20

NH3

R COOH

cop

NH2 -

t NH2

43

dihydropyrlmidinase

Fig. 2. Hydantoin

COO"

=R
R

HN

HN_xb

163-176

165

with dihydropyrimidinase, which catalyzes the cyclic ureide-hydrolyzing step of the reductive degradation of pyrimidine bases (Fig. 2B). The same results were obtained for other Pseudomonas species [22,23], Comamonas species [23], Bacillus species [9], Arthrobacter species [19], Agrobacterium species [22], and rat liver [24]. From these results, it is proposed that D-amino acid production from DL-5-monosubstituted hydantoins involves the action of the series of enzymes involved in the pyrimidine degradation pathway [ 19-211. However, this contention has remained moot because of the lack of systematic studies on the enzymes involved in these transformations [25,26]. We investigated the pyrimidine-transforming activity in typical hydantoin-transforming bacteria [27]. P. putida IF0 12996, which produces D-hydantoinase being identical with dihydropyrimidinase, naturally shows both D-hydantoinase and dihydropyrimidinase activity. Comamonas sp. E222c and Blastobacter sp. A17p-4, which have N-carbamoyl-D-amino acid hydrolyzing activity, show B-ureidopropionase activity. Blastobacter sp. also possesses both D-hydantoinase and dihydropyrimidinase activities. Thus, two cyclic ureide-hydrolyzing activities and/or two N-carbamoyl amino acid-hydrolyzing activities coexist in these bacteria (Table 1). However, the induction profiles of each coexisting enzyme activity for the several pyrimidineand hydantoin-related compounds did not correspond with each other in some cases (Table 2),

two enzyme-catalyzed reactions, the D-stereoOf DL-5-t phydrolysis specific hydroxyphenyl)hydantoin and subsequent hydrolysis of the D-carbamoyl derivative to D-phydroxyphenylglycine, is possible (Fig. 1). Based on these results, a new commercial process for the production of D-p-hydroxyphenylglycine has been developed [ 171. Enzymatic hydantoin cleavage is already important in the production of not only D- but also L-amino acids. Several microorganisms were found to have L-specific hydantoin-transforming activity. These microorganisms have been applied for the production of L-amino acids such as L-tryptophan and L-phenylalanine derivatives [ 181. In both D- and L-amino acids production from DL-Smonosubstituted hydantoins, the wide applicability to a broad substrate range together with their high stereoselectivity and their ability to convert completely a 100% hydantoin racemate into optically pure enantiomer render these processes very attractive.

(4

2 (1997)

(A) and pyrimidine

P_ureidoproplonase

(B) transformation

pathway.

J. Ogawa, S. Shimizu / Journal of Molecular Catalysis B: Enzymatic 2 (19971163-I

166 Table 1 Hydantoin-

and pyrimidine-transforming

Strain

Hydrolyzing

activities

in bacteria

activity to

cyclic ureide 5-monosubstituted P. putida IF012996 Comamonas sp. E222c Blastobacter sp. A17p-4

N-carbamoyl hydantoin

+ +

N-carbamoyl-Damino

+ -

-

4. Enzymes involved in IV-carbamoyl amino acid hydrolysis 4.1. N-Carbamoyl-D-amino acid amidohydrolase

To confirm the proposition described in the previous section, the enzymes hydrolyzing Ncarbamoyl-D-amino acids were first purified homogeneously from Comamonas sp. E222c [ 151 and Blastobacter sp. Al7p-4 [16], and characterized (Table 3). This enzyme is one of key enzymes in the production of D-amino acids from DL-5-monosubstituted hydantoins. The rel-

in growth media on hydantoin-

none dihydrouracil P-ureidopropionate DL-5-( p-hydroxyphenyl)hydantoin N-carbamoyl-Dp-hydroxyphenylglycine L DPase: Hydase: i UPase: DCase:

P-ureidopropionate + + +

ative molecular masses of the native enzymes and those of the subunits were approximately 120000 and 40000, respectively. These enzymes consist of three identical subunits. Both purified enzymes hydrolyzed various N-carbamoyl-Damino acids to D-amino acids, ammonia and carbon dioxide. N-Carbamoyl-Darnino acids having hydrophobic groups served as good substrates for these enzymes (Table 4). These enzymes strictly recognized the configuration of the substrate and only the D-enantiomer of the N-carbamoyl-o-amino acid was hydrolyzed. This property can be applied for the optical resolution of racemic N-carbamoyl-a-amino acids. These enzymes did not hydrolyze Bureidopropionate, suggesting that these enzymes are different from the enzymes involved in the pyrimidine degradation pathway, i.e., B-ureidopropionase. The substrates of known Ncarbamoylamide amidohydrolases were not hy-

and pyrimidine-transforming

Ratio of corresponding

acid

+ +

+

Compounds

amino acid

dihydropyrimidine

suggesting that these coexisting cyclic ureidehydrolyzing or especially N-carbamoyl amino acid-hydrolyzing activities are not always catalyzed by the same enzymes.

Table 2 Effects of various compounds

76

enzyme formation

enzyme activities

P. putida IF0 12996

Comamonas sp. E222c

Blastobacter sp. A17p-4

DPase “/Hydase

UPase ‘/DCase

DPase/Hydase

UPase/DCase

4.0 11.8 3.2 3.2 3.2

6.8 1.7 1.7 < 10-2 < 10-Z

18.7 16.9 16.9 17.4 18.9

dihydropyrimidine-hydrolyzing activity. 5-monosubstituted hydantoin-hydrolyzing activity. P-ureidopropionate-hydrolyzing activity. N-carbamoyk-amino acid-hydrolyzing activity.

b

2.7 2.1 5.1 3.3 5.1

d

167

J. Ogawa, S. Shimizu / Journal of Molecular Catalysis B: Enzymatic 2 (1 Y97) 163- 176 Table 3 Properties

of N-carbamoyl

amino acid-hydrolyzing N-Carbamoyl-D-amino

Properties

enzymes N-Carbamoylk-amino

P-Ureidopropionase

acid amidohydrolase

acid

amidohydrolase Comamonas SD.E222c native M, subunit M, (SDS-PAGE) number of subunits P’ Substrate specificity N-carbamoyl-a-amino N-carbamoyl+-amino N-carbamoyl-y-amino optimum pH optimum temperature pH stability thermal stability metal ion requirement

117000 40000 3 4.0 (stereospecificity) acid + b (D) _c acid _c acid

Blastobacter SD. Al7~-4

P. putida IF012996

Alcaligenes xylosoxidans

111000 40000 3 5.3

95000 44000 2 nd. ’

134000 65000 2 n.d. J

+ bCD)

+

+ bCL)

_c

+h +b

__c

h(L)

X.0-9.0

8.0-9.0

_c _c

7.5-8.2

8.0-8.3

40°C

55°C

60°C

35°C

7.0-9.0 < 40°C none

6.0-9.0 < 50°C none

6.0-8.5 < 65°C Co?’ Ni?+,Fe?+

6.0-9.5 < 30°C Co’+ Ni?+

Mn2+

,

.

Mn2i

a n.d.: not determined. b + Hydrolyzed. f - Not hydrolyzed.

Table 4 Substrate Compound

specificity

of N-carbamoyl-D-amino

( 10 mM)

acid amidohydrolases

Comamonas sp. E222c Relative activity (o/o)

Aliphatic N-carbamoylDalanine Dvaline Dleucine DL-alanine DL-o-amino-n-butyric acid DL-valine DL-norvaline DL-norleucine DL-methionine Aromatic N-carbamoylDphenylalanine Dphenylglycine D-p-hydroxyphenylglycine Dt-phenylalanine DL-tryptophan DLphenylglycine DL-p-hydroxyphenylglycine Others N-carbamoylDserine DL-serine DL-threonine * nd.: not determined.

23 10 28 20 14 8.4 6.3 90 92

100 24 47 59 55 14 39

7.2 6.7 3.0

Blastobacter sp. A17p-4 K, (mM)

Relative activity (so)

K, (mM)

12

29 55 60 27 48 50 26 92 84

4.0 0.41 0.36 n.d. ,’ n.d. A n.d. ” n.d. ’ 0.79 0.7 I

100 170 130 46 18 100 130

0.50 0.88 I .7 n.d. n.d. n.d. nd.

1.o 3.6 n.d. nd. nd. nd. 4.8 7.5

20 27 13 nd. n.d. n.d. n.d.

a * a ’

a a ’ a

24 nd. ’ nd. a

10

3.0 18

a ’ a a

2.4 nd. ’ n.d. A

J. Ogawa, S. Shimizu/Joumal

168

of Molecular Catalysis B: Enzymatic 2 (1997) 163-176

drolyzed by these enzymes, suggesting that they are novel N-carbamoylamide amidohydrolase. These enzymes were named as N-carbamoylD-amino acid amidohydrolase. The enzyme from Comamonas sp. was not significantly affected by N-carbamoyl+amino acid and ammonia, while the enzyme from Blastobacter sp. was inhibited by these products. Both enzymes did not require metal ions for the activity and were sensitive to thiol reagents. The enzyme from Blastobacter sp, was stable at high temperature (50°C) and suitable for the practical production of D-amino acids. 4.2. /3-Ureidopropionase

To make clear the difference between Ncarbamoyl-D-amino acid amidohydrolase and P-ureidopropionase, we purified and characterized the @_treidopropionase from aerobic bacteria for the first time [28]. The specific features of the purified enzyme from P. putidu IF0 12996 (Table 3) are quite different from those of the /3-ureidopropionases from other sources, such as mammals [29,30], protozoa [31] and

Table 5 Substrate Compound

specificity

of P-ureidopropionase

(10 mM)

anaerobic bacteria [32], and N-carbamoyl-Damino acid amidohydrolase in structure, in metal ion dependency and especially in substrate specificity. P-Ureidopropionase from P. putidu consisted of two identical polypeptide chains with relative molecular masses of 44000. The enzyme requires a divalent metal ion, such as Co*+, Ni*’ or Mn*+, for the activity. The enzyme showed a broad substrate specificity while the known @ureidopropionases are specific to N-carbamoyl-P-amino acids; not only N-carbamoyl+amino acids, but also Ncarbamoyl-y-amino acids, and several Ncarbamoyl-o-amino acids such as Ncarbamoylglycine, N-carbamoyl+alanine, Ncarbamoyl-L-serine, and N-carbamoyl-DL-aamino-n-butyrate are hydrolyzed (Table 5). NFormyl and N-acetylalanine are also hydrolyzed by the enzyme, but the rate of hydrolysis is lower than that for N-carbamoylalanine. The hydrolysis of N-carbamoyl-ar-amino acids is strictly L-enantiomer specific, i.e., the reverse stereospecificity to those of N-carbamoyl-Damino acid amidohydrolase and D-hydantoinase. This makes its difference from N-carbamoyl-D

from P. putida IF0 12996 Relative activity (%)

K, (mM)

V,,,

(pmol/min/mg)

V F m

N-Carbamoyl-P-amino acids N-carbamoyl+alanine (P-ureidopropionate) N-carbamoyl-x-@ninoisobutyrate N-Carbamoyl-y-amino acid N-carbamoyl-y-amino-n-butyrate N-Carbamoyl-a-amino acids N-carbamoylglycine N-carbamoyl-L-alanine N-carbamoyl+serine N-carbamoyl-m-u-amino-n-butyrate N-carbamoyl-Dr-norvaline N-carbamoyl-oL-threonine N-carbamoyl-oL-aspartate N-carbamoyl-L-asparagine N-carbamoyl+glutamate Others N-formyl-lx-alanine N-acetyl-DL-alanine

100 43

3.7 4.5

290

12

17 120 34 31 8.9 0.97 0.14 1.6 0.29

0.68 1.6 75 2.8 42 -

75 6.3

7.7 8.8

4.1 1.0 19 0.091 1.0 3.8 1.1 1.1

1.1 0.22 1.7 0.13 0.64 0.050 0.38 0.027

_ -

0.84 0.067

0.11 0.0077

169

J. Ogawa, S. Shimizu/ Journal of Molecular Catalysis B: Enzymatic 2 (1997) 163-176

enzyme showed a broad substrate specificity not only for short chain but also for long chain and aromatic N-carbamoyl-L-amino acids (Table 6). Furthermore, the enzyme did not hydrolyze pureidopropionate; suggesting that the enzyme is distinct from P-ureidopropionase in function. These results indicate that the enzyme is a novel specifically acting on Namidohydrolase carbamoyl-L-amino acids with broad substrate specificity. The enzyme was named as Ncarbamoyl-L-amino acid amidohydrolase. The broad substrate specificity and the strict stereospecificity make this enzyme applicable for optical resolution of racemic N-carbamoyl-olamino acids.

amino acid amidohydrolase clear, and is the reason why iV-carbamoyl-D-amino acid, which is produced by D-hydantoinase, is accumulated in P. putida IF0 12996. 4.3. N-Carbamoyl-L-amino acid amidohydrolase N-Carbamoyl-L-amino acid-hydrolyzing activities have been found in microorganisms [33371, and applied for the L-amino acids production from DL-Smonosubstituted hydantoins. To reveal whether these activities derived from pureidopropionase activity, we purified the enzyme hydrolyzing N-carbamoyl-L-amino acid from Alcaligenes xylosoxidans and characterized (Table 3) [38]. The enzyme from A. xyZosoxidans resembles P-ureidopropionase in structure (homodimer enzyme with subunits of 65000) and in metal ion dependency toward Mn2+, Ni2+ or Co2+ for activity. However, the substrate specificity of the enzyme was different from that of P-ureidopropionase. j3-Ureidopropionase from P. putida IF0 12996 hydrolyzed short chain ZV-carbamoyl-L-amino acids, but not long chain aliphatic and aromatic N-carbamoylL-amino acids [28]. On the other hand, the Table 6 Substrate Compound

specificity

of N-carbamoyl-L-amino

(10 mM)

N-Carbamoylamino

acid amidohydrolase

5. Enzymes involved in cyclic ureide hydrolysis 5.1. Diversity of cyclic ureide-hydrolyzing

In section 3, it is revealed that many kinds of enzymes with different stereospecificities and regiospecificities are involved in the N-

from A. xylosoxidans

Relative activity (%)

K, GM

15 100 64 28 24 9.1 12 5.3 4.5 19 8.8

6.6 3.0 1.5 0.40 0.85 0.86 3.2 0.31 0.92 10 -

V,,, (bmol/min/mg)

-“,,X K,

acids

N-carbamoylglycine N-carbamoyl-L-alanine N-carbamoyl-L-asparagine N-carbamoyl-L-valine N-carbamoyl-rx-norvaline N-carbamoyl-t_-leucine N-carbamoyl-w-methionine N-carbamoyl-L-isoleucine N-carbamoyl-L-phenylalanine N-carbamoyl-rx-serine N-carbamoyl-oL-threonine Others N-formyl-rL-alanine N-formyl-IX_-leucine N-formyl-rx-methionine N-acetylk-phenylalanine N-acetyl-Lx-norvaline

13 5.2 5.4 0.74 0.06

en-

zymes

16 -

1.9 4.1 1.2 1.4 0.95 0.16 0.23 0.080 0.070 0.81

0.28 1.6 0.80 3.4 I.1 0.19 0.072 0.26 0.076 0.081 -

0.47 -

0.029 -

J. Ogawa, S. Shimizu/.Joumal

170

of Molecular Catalysis B: Enzymatic 2 (1997) 163-176

Table 7 Properties

of cyclic ureide-hydrolyzing

Properties

enzymes

Cyclic ureide-hydrolyzing Blastobacter sp. A17p-4

enzymes from

N-Methylhydantoin amidohydrolase

dihydroorotase

fraction I (imidase)

fraction II (phydantoinase)

P. putida 77

P. putido IF0 12996

subunit M, (SDS-PAGE) number of subunits substrate specificity (stereospecificity)

105000 35000 3 cyclic imides dihydropyrimidines

200000 53000 4 dihydropyrimidines 5-monosubstituted hydantoins (D)

290000 70000 and 80000 cx2p2 N-methylhydantoin 5-monosubstituted hydantoins (L)

82000 41000 2 dihydroorotate dihydroorotate methyl ester

Optimum pH for ring-opening hydrolysis cyclicizing dehydration optimum temperature pH stability thermal stability metal ion requirement

7.5-8.0 6.5 60°C 6.0-9.0 < 60°C (coZ+ )

9.0- 10.0 5.0 60°C 5.0-8.5 < 60°C activation (Ni*+, Co”,

8.0 (not catalyzed) 37°C 5.5-8.5 < 40°C Mg2+ and K+

8.5-9.0 4.5-5.0 60°C 6.0-9.0 < 55°C Zn*+

native kf,

carbamoyl amino acid hydrolysis. These results suggest that diverse enzymes may also be involved in catalysis of the upstream reaction, cyclic ureide hydrolysis. Table 2 shows, in Bhstobacter sp., more than two enzymes under the different induction control are involved in cyclic ureide hydrolysis. Further purification of these enzymes proved the occurrence of two cyclic ureide-hydrolyzing enzymes in this bacterium. Two dihydrouracil hydrolyzing fractions

Fig. 3. Substrate specificities

of the two cyclic ureide-hydrolyzing

(L)

Mn’+)

(fraction I and II> were obtained by phenyl-Sepharose CL4B column chromatography. These two fractions showed many differences in enzymological properties (Table 71, induction profiles and substrate specificities [39]. Fraction I preferably hydrolyzed cyclic imide compounds such as glutarimide and succinimide more than cyclic ureide compounds such as dihydrouracil and hydantoin (Fig. 3). The reaction product derived from succinimide was iden-

enzymes (fraction I and II) from Blastobacter

sp. A17p-4.

J. Ogawa, S. Shimizu/ Journal of Molecular Catalysis B: Enz~vmaric 2 (1997) 163-l 76

0

F

d H&

-

o

f+carbamoylearcosine

Nmethylhydantoin

Fig. 4. N-Methylhydantoin

amidohydrolase

tified as succinamic acid. Thus, fraction I catalyzed the hydrolysis of cyclic imides to monoamidated dicarboxylates stoichiometritally. Because there have been no reports on the enzyme which shows same substrate specificity as that of fraction I, it is considered to be a novel enzyme, which should be called an imidase. Fraction II hydrolyzed dihydropyrimidines and 5substituted hydantoins to the corresponding N-carbamoyl amino acids, but not cyclic imides (Fig. 3). The hydrolysis of 5-substituted hydantoins was o-stereospecific, because the products, N-carbamoyl amino acids, were hydrolyzed by N-carbamoyl-D-amino acid amidohydrolase but not by N-carbamoyl-L-amino acid

Table 8 Substrate

specificity

of N-methylhydantoin

Substrate

COOH NH2 + ADP + Pi c N--1( HBi= o

Mg*+, K+ NH+ATP+2H20

amidohydrolase

catalyzed reaction.

amidohydrolase. This bacterium has both Ncarbamoyl-D-amino acid amidohydrolase and N-carbamoyl-L-amino acid amidohydrolase. The reason why only o-amino acids are produced from DL-5monosubstituted hydantoins by this bacterium is the strict stereospecificity of fraction II to o-5-monosubstituted hydantoins. 5.2. N-Methylhydantoin L-hydantoinase

amidohydrolase

An ATP-dependent amidohydrolase, N-methylhydantoin amidohydrolase, which catalyzes the reaction presented in Fig. 4, was first found in Pseudomonas putida 77 by us [40,41]. The enzyme catalyzes the second step reaction in the

from P. putida 77 for amide compounds

and pyrimidines

Products found Cwbamoyl

amino acid

ADP

K,

“I,,

(pmol/ml)

(mM)

(pmol/min/mg)

N-methylhydantoin hydantoin t%%methylhydantoin glutarimide succinimide 2-pyrrolidone 2-oxazolidone F-valerolactam 2,4_thiazolidinedione 2Gmidazolidone L-5-oxoproline methyl ester DL-5-oxoproline methyl ester

4.03 1.15 0.93 1.91 2.5 1 n.h. a n.h. a n.h. a n.h. a n.h. a nh. ’ n.h. ’

dihydrouracil dihydrothymine uracil thymine

nh. n.h. n.h. n.h.

4.33 1.20 1.02 4.34 4.38 3.87 1.20 2.22 2.92 1.95 0.55 0.74 3.87 2.92 3.17 1.31

0.032 1.7 2.0 0.070 0.13 0.75 18 0.11 10 40 n.d n.d. b 0.083 2.0 6.6 16

9.0 3.6 3.1 2.2 2.2 4.1 2.1 2.4 3.8 3.1 nd. ’ n.d. h 5.8 4.3 8.2 2.8

a a a a

with

activity

(pmol/ml>

a n.h. = not hydrolyzed. ’ n.d. = not determined.

171

172

J. Ogawa, S. Shimizu / Journal of Molecular Catalysis B: Enzymatic 2 (1997) 16% I 76

degradation route from creatinine to glycine, via N-methylhydantoin, N-carbamoylsarcosine, and sarcosine as successive intermediates [40-481. N-Methylhydantoin amidohydrolase resembles dihydropyrimidinase in that both enzymes hydrolyze hydantoin compounds. Different from dihydropyrimidinase, however, N-methylhydantoin amidohydrolase requires ATP for the hydrolysis of its substrates. The enzyme is inducible only with the presence of creatinine and N-methylhydantoin, suggesting that the role of this enzyme is in the transformation of creatinine [49]. This enzyme was purified to homogeneity from P. putidu 77 and characterized (Table 7) [49]. The enzyme has a relative molecular mass of 290000. It is a tetramer of two identical small subunits (relative molecular mass of 70000) and two identical large subunits (relative molecular mass of 80000). The enzyme requires ATP for the amidohydrolysis of N-methylhydantoin and vice versa. Mg2+, Mn2+ or Co2+, and K+, NH:, Rb+ or Cs+ were absolutely required concomitantly for the enzyme activity as divalent and monovalent cations, respectively. The hydrolysis of amide compounds and coupled hydrolysis of ATP were observed with hydantoin, L-5methylhydantoin, glutarimide and succinimide besides N-methylhydantoin (Table 8). Some naturally-occurring pyrimidine compounds such as dihydrouracil, dihydrothymine, uracil and thymine, effectively stimulate ATP hydrolysis by the enzyme without undergoing detectable hydrolysis of themselves (Table 8). Furthermore, the enzyme exhibits unique nucleoside triphosphatase activity [50]. Besides ATP, various kinds of nucleoside triphosphates can substitute and be hydrolyzed to yield their corresponding nucleoside diphosphates not only in the presence but also in the absence of amide compounds. The ATP-dependent hydrolysis of 5monosubstituted hydantoins, for example 5methylhydantoin, by the enzyme proved to be L-isomer specific [49], because the produced N-carbamoylalanine was hydrolyzed by Ncarbamoyl-L-amino acid amidohydrolase but not

by N-carbamoyl+amino acid amidohydrolase. These results indicate that such a kind of enzyme is applicable for the L-amino acid production from DL-5monosubstituted hydantoin. 5.3. Dihydroorotase Dihydroorotase (EC 3.5.2.3) is one of the known cyclic ureide-hydrolyzing enzymes, which catalyzes the reversible cyclization of L-ureidosuccinate to dihydro-L-orotate, the third step in pyrimidine biosynthesis. Given the information provided by comparative studies on the function, structure and evolution of cyclic ureide-hydrolyzing enzymes, dihydroorotase from P. putida IF0 12996 was purified to homogeneity and characterized (Table 7) [51]. The relative molecular mass of the native enzyme was 82000 and the enzyme consisted of two identical subunits with a relative molecular mass of 41000. The enzyme only hydrolyzed dihydro-L-orotate and its methyl ester, and the reactions were reversible. These results make clear the difference of dihydroorotase from dihydropyrimidinase. Dihydroorotase is specific for the sixmember cyclic ureides and shows the stereospecificity for the L-isomer. The enzyme was inhibited non-competitively by pyrimidinemetabolism intermediates such as dihydrouracil and orotate, with Ki values of 3.4 and 0.75 n&I, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays some role in the control of pyrimidine biosynthesis.

6. Application of microbial hydantoin-transforming enzymes Microbial hydantoin-transforming enzymes have been used for the production of optically active amino acids from DLJ-monosubstituted hydantoins. A variety of enzymes have been reported on by other workers, Runser et al. [52] reported the occurrence of D-hydantoinase with-

1. Ogawa,

S. Shimizu/Joumal

of Molecular

Catalysis

.D-Hydantohaae with dihydmpyrimidinase

etc.

for D-hydantom

Agmbacfefium

?? ATP-independent Flavobacterium

Affhrobacfer

?? ATP-dependent

Pseudomonas

?? L-Hydantolnaaa

?? ATP-independent t?&f/US

Alfhrobacter etc

?? ATP-dependent BaCillUi

?? N Mathylhydantoin amldohydrdaaa ATPdependentand specificfor L-hydantom Pseudomonas

active amino acid production

processes

out dihydropyrimidinase activity. Watabe et al. [53] reported that ATP-dependent hydantoin hydrolyzing enzyme is involved in the L-amino acid production from DL-Zkmonosubstituted hydantoin by Pseudomonas sp. NS671. This enzyme shows no stereospecificity. Hydantoinase showing no stereospecificity and not requiring ATP was also reported [54]. Furthermore, recently, hydantoin racemizing enzymes were

using hydantoin-transforming

4

D-Amino acid

4

L-Amino acid

enzymes.

Pyrimidlna transformation pathway

COOH

COOH

+ Hz0 -H’xN%

+

+ H20

NH3 + C4

-

c

+

NH3 + CC+

+

NH3 + CC+

+

NH3 + CC+

NH;! COOH + H20

-

c NY

H H;! + H2” R

Fig. 6. Reactions

catalyzed

by N-carbamoyl

1

found [55,56]. This racemase is able to totally convert racemic substrate, which only slowly racemizes under reaction conditions, to single stereoisomer. The combinations of these hydantoin-transforming enzymes provide a variety of processes for the production of optically active amino acid (Fig. 5). In addition, IV-methylhydantoin amidohydrolase is also useful for diagnostic use. N-Methyl-

Hydantoln tranaformatlon pathway

R

173

76

acid amldohydrolaae Comamonas Blastcbacter Agrobacterium etc. ?? NCarbamoylaarcoaina amldohydrolaaa D-specificfor Ncarbamayl. a-aminoacids Pseudomonas ?? NCarbamoyl-L-amino acid amldohydrolaaa Alcaliganes Navcbacterium Arthrcbatier etc. ?? B_Uraldoproplnaaa L-specificfor karbamoyla-aminoacids Pseudomonas ?? Chemical dacabamoylation

?? DL-Hydantolnaae

Fig. 5. Optically

163-l

,?? N-Carbamoyl-D-amlno

?? identical

?? specific

2 (1997)

KCarbamoyl amino acid hydrolysis

Cyclyc ureide hydrolysis

Pseudomonas i%Wobacter

B: Enzymatic

amino acid-hydrolyzing

enzymes

J. Ogawa, S. Shim&/

174

N-Carbamoyl-L-amino acid amidohydrolase

\

p-Ureldopropionase Fig. 7. Comparison

of N-terminal

Journal of Molecular Catalysis B: Enzymatic 2 (1997) 163-176

A.

xylosoxidans

P.

put/da

P

/ MTPAQQVLQSTQIiHIDSTRL

amino acid sequence of phydantinase

from Blastobacter

sp. A17p-4 with those from Pseudomonas and

Bacillus species [ 171.

hydantoin amidohydrolase and the successive enzyme in microbial creatinine transformation, i.e., N-carbamoylsarcosine amidohydrolase, are useful for the enzymatic measurement of creatinine in serum and urine which is a marker of renal dysfunction [41]. This enzymatic method has proved to be simple and precise, and has excellent sensitivity and specificity [57].

7. Discussion This review describes the enzymes involved in the microbial transformations of hydantoinrelated compounds. These transformations have been applied for the production of optically

active amino acids from DL-5monosubstituted hydantoins [ 1858,591. These transformations generally involve ring-opening hydrolysis of cyclic ureides and subsequent decarbamoylating hydrolysis of N-carbamoyl amino acids. A variety of enzymes are involved in these two reactions. In the hydrolysis of iV-carbamoyl amino acid, three types of enzymes with different substrate specificity function (Fig. 6). Two types are specific for N-carbamoyl-cx-amino acids, but show reverse stereospecificity. These two enzymes, &Xii-bamoyl-D-amino acid amidohydrolase and acid amidohydrolase, N-carbamoyl-L-amino contribute to the production of D-amino acids or L-amino acids from DL-5-monosubstituted hy-

AkMethylhydantoin Fig. 8. Reactions catalyzed

by cyclic ureide-hydrolyzing

amidohydrolaae

enzymes.

J. Ogawa,

S. Shimizu/

Journal

of Molecular

dantoins, respectively. The other type of enzyme, /3ureidopropionase, shows broad specificity for N-carbamoyl-a-, iV-carbamoyl+and N-carbamoyl-y-amino acids, and functions in the pyrimidine metabolism. p-Ureidopropionase hydrolyzes short chain N-carbamoyl-o-amino acids L-stereospecifically. This specificity is the same as N-carbamoyl-L-amino acid amidohydrolase. These two enzymes show similarities in dimer structure, and metal ion dependence. However, the N-terminal amino acid sequences of these two enzymes are quite different. It resembles that of the N-carbamoyl-D-amino acid amidohydrolases (Fig. 7). In cyclic ureide hydrolysis, as well as in N-carbamoyl amino acid hydrolysis, various enzymes are concerned (Fig. 8). D-Hydantoinase, which is regarded to be identical with dihydropyrimidinase, catalyzes the first step of Damino acid production from DL-5-monosubstituted hydantoin. This kind of enzyme has been reported in some other bacteria and mammals, and all of them preferably hydrolyze dihydropyrimidines. They show strong similarity in their N-terminal amino acid sequences (Fig. 9). We found another enzyme which hydrolyzes dihydropyrimidines and more actively acts upon the cyclic imides. Furthermore, Runser et al. [52] reported the occurrence of D-hydantoinase without dihydropyrimidinase activity. These findings indicate the diversity of cyclic ureidehydrolyzing enzymes. For L-amino acid production, the ATP-dependent enzyme, N-methylhydantoin amidohydrolase, is applicable. This enzyme originally functions in the creatinine degradation. Recently, Watabe et al. [53] reported that ATP-dependent hydantoin hydrolyz-

BlaatobtKtef sp.A17p-4 P. @da IF0 12996 P. tluon9.9ccns OSM 04 Elasfobacter sp. Al 7~4 Bacillus LUl220 8aClllUS CB!636360

Fig. 9. N-Terminal amino acid sequences acid-hydrolyzing enzymes.

of IV-carbamoyl

amino

Catalysis

B: Enzymatic

2 (1997) 363-176

175

ing enzyme is involved in the L-amino acid production from DL-5monosubstituted hydantoin by Pseudomonas sp. NS671. This enzyme is different from N-methylhydantoin amidohydrolase in that it shows no stereospecificity and does not hydrolyze N-methylhydantoin, but is similar to N-methylhydantoin amidohydrolase especially in N-terminal amino acid sequence [49]. Another enzyme showing L-stereospecificity is dihydroorotase, which hydrolytically produces N-carbamoyl-L-aspartate from dihydroorotate. However, this enzyme is specific for six-membered cyclic ureides, dihydropyrimidine, and does not act upon the five membered cyclic ureides, hydantoin. Above all, a variety of enzymes is involved in microbial hydantoin transformation and shows versatile ability. One of their superior ability is stereospecificity which is applicable to the production of optically active compounds. Other than this, N-methylhydantoin amidohydrolase is also useful for diagnostic use. As we presented here about microbial transformation of hydantoin-related compounds, the specific feature of the microbial world is its unusual diversity. Application of microbial hydantoin transformation is one good example of the high value of diverse microbial world.

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