D-AMINO ACID RESEARCH: MY RETROSPECT AND PERSPECTIVE
Kenji Soda,
Department of Biotechnology, Kansai University
Yamatecho, Suita, Osaka-fu 611-0002, Japan
Fax; 0774-32-7049(Received

E- mail; soda@okurayama.mbox.media.kyoto-ac.jp

12 January, 2006 Accepted 20 February, 2006)

mammalian tissues or fungi to prepare L-amino

Introduction
D-Amino acids were once regarded as
unnatural compounds, but that is not the case.

Itacids.

D-Aminoproducts.

acids were produced as side-We

firstusing

microbial

is now highly probable that D-amino acids

aminotransferasesprebiologically occurred in the primordial soup.

prepared, and found a few new aminotransferases

The L-amino acids of the racemates synthesized onsuch as L-lysine ε -aminotransferase(5) andthe primeval Earth were selectively incorporated

taurineinto proteins leading to the production of enzymes

screening the enzymes participating in D-amino

and eventually the birth of the first organisms.

acid metabolism as well.

Accordingly, the first organisms were quite likely

account of my biochemical research on D-amino

emerged in the presence of racemic or D-aminoacids with a perspective.

acids.

bystudiedaminotransferse(6).

L-aminoWeacids

undertook

I describe here a brief

In fact, D-amino acids are found in bothfree and petidyl forms in hyperthermophilic

Results and Discussion
We found that various species of Bacillus

archaea, which are presumed to be the mostprimitive organisms.

Various D-amino acids areabundantlyproduce

D-aminoacid

now demonstrated not only microorganisms, butaminotransferase, and purified and characterizedalso in higher plants, insects, marine invertebrates

the enzymes from B. sphaericus(7) and a moderate

and mammals (1-3). Only recently, it was reported

thermophile, Bacillus

sp. YM-1 isolated from athat D-histidine and D-lysine react with respective

dustTheaminoacyl-tRNA synthetases of Escherichia coli

thermophile is thermostable.

(4) : D-amino acids may be incorporated into usualstructure,proteins to produce unusual proteins, though verymechanism, suicide-substrate reaction mechanism

slowly.

and stabilization mechanism by using the enzyme

In the late 1950’s when we began to study

ofsauna(8).

enzymologicalenzymefromtheWe showed theproperties,

reaction

produced by the clone cells of E. coli (9).

TheD-amino acid biochemistry, only a few scientists

stereospecificity of enzyme is absolute: D-amino

were interested in D-amino acids, and we wereacids are the exclusive amino donors and theengaged in enzymological study of microbial L-

products from the amino acceptors.

amino acid aminotransferases.

Only several

structural specificity is very low: a variety of D-kinds

commercially

amino acids and 2-oxo acids are the amino donorsof L-amino acids wereavailable,and expensive.

Accordingly,weandacceptors,respectively.

However, theTheprimarysynthesized the racemates chemically, and resolve

structure of D-amino acid aminotransferase isthem by means of aminoacylase isolated fromsignificantly homologous to that of branched-chain

Viva Origino 34 (2006) 81 - 85
ⓒ 2006 by SSOEL Japan- 81 -

Viva Origino 34 (2006) 81 - 85

L-amino acid aminotransferase of E. coli, though

re-faceboth enzymes fall into the same subgroup ofaminotransferase, in which the residues are shownaminotrqnsferases on the basis of the primaryon the si-face side(12).

structure, and show no similarity to all otheraminotransferase is peculiar in the substrateaminotransferases(10).

specificity,The

circulardichroism

sideinthecontrast

toaspartate

Thus, D-amino acidprimarystructure,

thespectra of various L-amino acid aminotransferases

stereostructure of the active center and thesuch as aspartate aminotransferase show positive

stereospecificity for the hydrogen transfer.

bands at the wavelengths where their internal

Amino acid racemases are unique in theSchiff base intermediate absorbs, but that of D-substrate stereospecificity and mode of the reaction.

amino acid aminotransferase shows the negative

They apparently catalyze a simple reaction, namely

band at 430 nm.

theremovalofα -hydrogenfromeither

proceeds

enantiomer of a substrate amino acid, andthrough an external Schiff base (Fig. 1). D-Amino

subsequently a non-stereospecific reprotonationacid aminotransferase and branched-chain L-amino

occursacid

uniqueenantiomers. Almost all the enzymes catalyze

stereospecificity of hydrogen transfer between C-1

various reactions with only one of an enantiomericof the amino acid moiety ofand diastereomeric pair of chiral compounds, butTheaminotransferaseaminotransferase

reactionshow

thealdimine Schiff basetoattainequilibrium

betweenbothintermediate and C-4’ of the coenzyme moiety of

the racemases lack the stereospecificity.

ketimine Schiff base intermediate through thethem require pyridoxal phosphate as a coenzyme,

carbanionic intermediate.

Both catalyze the pro-but some others such as glutamate racemase andR specific hydrogen transfer in contrast to otherproline racemase require no coenzymes and no

aminotransferasesaminotransferase

asaspartate

metals:ornithineδ-requirement.

The

practicallysuchandaminotransferase as shown in Fig. 2
three-dimensional

structureaminotransferase is peculiar.

of(11).

D-aminoThe catalyticallytheyarediversein

theMost ofcoenzyme

Almost all the racemases show
theabsolutesubstratespecificity.

Glutamate racemase acts exclusively on D- and Lglutamates.

In contrast, arginine racemase andimportant amino acid residues including theamino acid racemase with low substrate specificity

internal Schiff base lysine 145 are located on thefromFig. 1.

Pseudomonas

bacteria

Reaction Mechanism of Aminotransferase (Half-reaction)- 82 -

foundinourViva Origino 34 (2006) 81 - 85

Fig. ２. Stereochemistry of Hydrogen Transfer by Aminotransferase.
AspAT; Aspartate Ainotransferase
OAT; Ornithineδ-Aminotransferase AlaAT;
Alanine Aminotransferase DAAT; D-Amino Acid Aminotransferase
BCAT;
Branched-Chain Aminotransferase

laboratory catalyze the racemization of a variety ofpoly- γ -glutamate in abundance, which is aamino acids except for acidic and aromatic amino

polycation composed of D-glutamate and L-acids (13). We showed that the hydrogenisglutamate with the molar ratio of about 80:20.

between theThe ratio depends on the species and the growthtransferred

non-stereospecificallyamino acid C-1 of the aldimine Schiff baseconditions.

intermediate and the coenzyme C-4’ of theisozymes of glutamate racemase, Glr and YrpC.

ketimineintermediate

intheWe showed the occurrence of two

transamination

Glr participates in the poly-γ-glutamate synthesis,

catalyzed as a side reaction by alanine racemase

and YrpC is produced only on cell division, and(Fig 2).

involved in the synthesis of petidoglycan of cellwall by a supply of D-glutamate, an essential

Glutamate racemase is a typical coenzymeindependentaminoacidracemase.

Weconstituent.
Poly- γ -glutamatecharacterized the racemase from a lactic acidsynthetasecatalyzes

bacterium, Pediococcus pentosaceus

synthesis of the polymer from D- and L-glutamates(14). The enzyme consists of a single subunit within the presence of ATP(15).

molecular weight of about 29,000, and contains angroup of a C-terminal glutamate residue of theessential cysteine residue at the active center,growing poly- γ -glutamate is first activated towhich probably serves as a catalytic base toform a phosphate ester with hydrolysis of ATP.

abstract the substrate α-hydrogen.

It shows noAn amino group of a substrate glutamate attacks

structural similarity to other enzymes, but thethe activated carboxyl group as a nucleophile tostructure of active center is closely similar to theelongate the polymer: this is an ATP andheme binding site of hemoglobin and myoglobin.

glutamate-dependent γ -ligase reaction.

Hemine stoichiometrically binds to the enzyme

showed that poly-γ-glutamate synthetase consists

active center to inhibit the activity.

of three components. Component PgsB, which isthe producers of Japanese and Koreanfermented

Chungkookjang,food,respectivelyWefound inside the cell membrane, catalyzes the ATP

Several species of B. subtilis, in particularsoybeanThe γ -carboxyland

Nattoandproduce

viscousglutamate-dependent

probablyinassociation

γ -ligase
withPgsC.

reaction
Thiscomponent occurs in the hydrophobic region of- 83 -Viva Origino 34 (2006) 81 - 85

Component PgsA is not involved

as a sweetener or a medicine in bound forms, if

in the poly-γ-glutamate chain elongation directly,

more effective methods for production of D-amino

but severs as a kind of transporter, and protects theacids are developed and industrialized.

polymer formed from the enzymatic and non-research of molecular evolution of D-amino acid -enzymatic depolymerization.

metabolizing enzymes and D-amino acids also willcell membrane.

reaction is peculiar.

The synthetase

The D,L- ratio of the poly-γ

Thestimulate biochemistry and molecular biology.

-glutamate is not regulated by the enzyme, butdepends on the D,L-ratio of substrate glutamate,

References

which is controlled by glutamate racemase Glr.

(1) Meister, A. (1965) Biochemistry of the Amino

The three dimensional structure of synthetase andAcids(2ndedition)113-118,

the detailed reaction mechanism have not yet been

Academic Press.

York:(2) Jolles, P. (ed) (1998) D-Amino Acids inelucidated.

Sequencesof

SecretedPeptidesof

Multicellular Organisms, Basel, Birkhauser

Perspective of the Future

VerlagGreat strides have been made in D-amino(3) Fujii, N., Saito, T., (2004) Homochirality andacid research since I was first engaged in theresearch.

Newlife. Chem Rec 4, 267-278

However, we are not yet systematic in

the biochemistry of D-amino acids in comparison(4) Takayama, T., Ogawa, T., Hidaka, M., Shimizu,

with that of L-amino acids for various reasons. ForY., Ueda, T., Masaki, H. (2005) Esterification

example, analytical techniques for the specific andof Eschericia coli tRNAs with D-histidine andaccurate determination of D-amino acids have not

D-lysine

been fully advanced. The development of effectiveBiosci. Biotechnol. Biochem. 69, 1040-1041

analytical methods for free and bound D―amino(5) Soda, K., Misono,H. (1968) L-Lysine- α -byaminoacyl-tRNAsynthetases.

acids will lead to elucidation of the occurrence andKetoglutaratemetabolism of D-amino acids in various organisms.

Purification, Crystallization and Properties.

Only several kinds of enzymes involved in D-Biochemistry, 7, 4110-4119

Aminotransferase.

II.

amino acid metabolism have been studied in detail,(6) Toyama, S., Soda,K., (1972) Occurrence ofand various other important enzymes such as D-Taurine: α -Ketoglutarate Aminotransferae inglutamate cyclase, D-amino acid dehydrogenase

Bacterial Extracts. J. Bacteriol. 109, 533-538

and hydoxyproline epimerase have been little(7) Yonaha, K., Misono, H., Yamamoto, T.,

investigated.

The detailed information of D-Soda,K.,(1975)

amino acid metabolizing enzymes will result inAminotransferae

ofelucidation

Enzymologicalandofmetabolic

functions of D-amino acids.

andphysiologicalThe easy availability

D-AminoAcidBacillus

sphaericus:

Spectrophotometric

Properties. J. Biol. Chem. 250, 6983-6989

of pure D-amino acids uncontaminated with the L-(8) Tanizawa, K., Masu,Y., Asano, S., Tanaka, H.,

enantiomers is also desired to get the accurate

Soda, K., (1989) Thermostable D-Amino Acid

information of D-amino acid biochemistry.

Aminotransferase

The elucidation of physiological roles of D-a

Thermophilic

Bacillus Species. J. Biol. Chem. 264, 2445-

amino acids will open a new field of bioscienceand biotechnology.

from2449

Some of D-amino acids such(9) Sugio, S. Petsko, G. A., Manning, J. M., Soda,

as D-tryptophan taste very sweet, and may be used

K., Ringe, D. (1995) Crystal Structure of a D-- 84 -Viva Origino 34 (2006) 81 - 85

Amino Acid Aminotransferase:How the Proteinand Properties. J. Biol. Chem., 246, 5085-5092

Controls Stereospecificity. Biochemistry, 34,(13) Soda, K.,Osumi, T. (1969) Crystalline Amino
Acid Racemase with Low Substrate Specificity.

9661-9669(10) Tanizawa, K., Asano,S., Masu., Kuramitsu, S.,

Biochem. Biophys. Res. Commun. 35, 363-366

Kagamiyama, H., Tanaka, H. Soda.,K. (1989)(14) Choi, S. –Y., Esaki, N., Yoshimura, T.,

The Primary Structure of Thermonstable D-Soda,K. (1991) Overproduciton of Glutamate

Amino

Aminotransferase

froma

Racemase of Pediococcus pentosaceus in

ThermophilicBacillus

SpeciesandIts

Escherichia

CorrelationwithL-Amino

AcidPurification.

Acidcoli

Clone

Protein

CellsandExpression

ItsandPurification 2, 90-93

Aminotransferase. J. Biol. Chem.264, 2450-(15) Ashiuchi, M., Soda, K., Misono, H. (1999) a2454(11) Yoshimura T., Nishimura, K., Ito, J., Esaki, N.,

Poly-γ-glutamate synthetic system of Bacillus

Kagamiyama, H., Manning, J.M., Soda, K.

subtilis(1993) Unique stereospecificity of D-amino

Biochemical Analysis of Poly- γ -glutamateacid aminotransferase and branched-chain L-

produced by Eschrichia coli clone cells.

amino acid aminotransferase for C-4’hydrogen

Biochem. Biophys. Res. Commun. 263, 6-1

transfer of the coenzyme. J. Amer. Chem. Soc.
115. 3897-3900(12) Yorifuji, T., Misono, H., Soda, K., (1971)
Arginine

RacemaseofPseudomonas

graveolens. Part I. Purification, Crystallization- 85 -IFO

3336:

Gene

Cloningand