- Email: [email protected]
Branched-Chain Amino-Acid Aminotransferase of Escherichia coli
[11]
E. £oli BRANCHED-CHAINAMINOTRANSFERASE
103
now also been identified, 14-18 and we expect that reconstitution titrations with the catalytic subunits from the same organisms will become important tools in their biochemical characterization. We should also point out the usefulness of isolated A H A S subunits in the study of the physiology of branched-chain amino acid biosynthesis. Because of the tendency of at least some of the A H A S isozymes to dissociate on dilution, the intracellular activities of these enzymes may be underestimated when they are assayed. 7,19 We have used the addition of small subunits to the assay mixture to correct this p r o b l e m ) 9 If, as now seems likely, eukaryotic A H A S s also have a labile quaternary structure, the addition of the appropriate small subunits would also help clarify the real physiological activity of these AHASs.
Acknowledgment T h i s w o r k w a s s u p p o r t e d in p a r t b y G r a n t 243198 f r o m t h e I s r a e l S c i e n c e F o u n d a t i o n .
14N. Ohta, J. Plant Res. 110, 235 (1997). 15D. Chipman, Z. Barak, and J. V. Schloss, Biochim. Biophys. Acta 1385, 401 (1998). 16p. Bork, C. Ouzounis, C. Sander, M. Scharf, R. Schneider, and E. Sonnhammer, Protein Sci. 1, 1677 (1992). 17R. (3. Duggleby, Gene 190, 245 (1997). 18M. E. Reith and J. Munholland, Plant Mol. Biol. Rep. 13, 333 (1995). 19S. Epelbaum, R. A. LaRossa, T. K. VanDyk, T. Elkayam, D. M. Chipman, and Z. Barak, J. Bacteriol. 180, 4056 (1998).
[ 1 11 B r a n c h e d - C h a i n
Amino-Acid Escherichia
Aminotransferase
of
coli
B y HIROYUKI KAGAMIYAMA and HIDEYUKI HAYASHI
Ovemew In bacteria, branched-chain amino-acid aminotransferase (BCAT) catalyzes the last step of biosynthesis of branched-chain amino acids. Valine, leucine, and isoleucine are synthesized from the corresponding keto acids
METHODS IN ENZYMOLOGY, VOL. 324
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 0076-6879/00 $30.00
104
ENZYMECLONING,EXPRESSION,AND PURIFICATION
[1 1]
by amino-group transfer from glutamate. In Escherichia coli, B C A T is encoded by the gene ilvE, which is a component of the i l v G E D A operon. Earlier studies of E. coli transaminases indicated that there are two kinds of transaminase activities. Transaminase A is active toward dicarboxylic amino acids, and transaminase B is active toward branched-chain amino acids. 1 Transaminase A was later resolved into two enzymes: one has narrower specificity for dicarboxylic amino acids, and the other has broader specificity and catalyzes the transamination of aromatic amino acids as well as dicarboxylic amino acids. The first, high-specificity enzyme was identified as the E. coli counterpart of aspartate aminotransferase (AspAT), which is prevalent throughout species and is the most abundant and best-studied aminotransferase. 2,3 The second enzyme was determined to be an aromatic amino acid aminotransferase, and has an important role in catalyzing transamination between glutamate and aromatic keto acids, the last step in the biosynthesis of phenylalanine and tyrosine. 2'3 Transaminase B was clearly distinguished from these aminotransferases by the absence of activity toward aspartate. Structural bases for this difference have been provided both in primary and in three-dimensional structures, and are summarized in this chapter. A s s a y Method Principle
The enzymatic transamination between branched-chain amino acids and 2-oxoglutarate can be followed by measuring the production of glutamate through coupling to the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, using glutamate dehydrogenase, NAD, and 1-methoxy-5-methylphenazinium methosulfate. 4 In the original procedure, 5 diaphorase and 2-(p-iodophenyl)-3-(p-nitropheny)-5-phenyltetrazolium chloride were used in place of 1-methoxy-5-methylphenazinium methosulfate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide. Procedure
The assay mixture contains 50 m M H E P E S - N a O H (pH 8.0), 0.1 M KC1, 10 m M isoleucine, 10 m M 2-oxoglutarate, 2.5 m M N A D (oxidized 1D. Rudman and A. Meister, J. Biol. Chem. 200, 591 (1953). 2 D. H. Gelfand and R. A. Steinberg, J. Bacteriol. 130, 429 (1977). 3j. T. Powell and J. F. Morrison, Eur. Z Biochem. 87, 391 (1978). 4 K. Inoue, S. Kuramitsu, T. Ogawa, H. Ogawa, and H. Kagamiyama, J. Biochem. 104, 777 (1988). 5R. Rej, Anal. Biochem. 119, 205 (1982).
[i I]
E. coli BRANCHED-CHAINAMINOTRANSFERASE
105
form), 30 tzM 1-methoxy-5-methylphenazinium methosulfate, 0.2 mM 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, and 0.2 mg of glutamate dehydrogenase, in a total volume of 1.0 ml. The reaction is started by the addition of enzyme, and the absorbance at 500 nm is continuously monitored. The molar extinction coefficient is determined by measuring the absorbance change on addition of known amounts of glutamate to the assay mixture. The value is generally about 2 X 104 M -~ cm -a. Transamination between other amino acids and 2-oxoglutarate can be measured by substituting isoleucine with other amino acids. Cloning of ilvE Gene The chromosomal D N A of E. coli W3110 is prepared according to a standard method, 6 and digested partially with Sau3AI. The D N A fragments of about 1000 base pairs (bp) are collected on a sucrose density gradient] and ligated to the BamHI site of pBR322. The plasmid is used to transform E. coli AB2227 (ilvE-). The transformants that suppress the branchedchain amino acid auxotroph are selected on minimum agar plates supplemented with ampicillin (50/~g ml-1). The complete nucleotide sequence of the ilvE gene with its neighborhood and deduced amino acid sequence is shown in Fig. i. Purification Procedures The methods for overproduction and purification of BCAT are modified from the previously published methods. 4 Overproduction The cloned ilvE gene is excised at the SalI and X m n I sites flanking the gene, and is ligated to the SalI-SmaI site of pUCll9. The resultant plasmid, named pUCll9-ilvE, is used to transform E. coli JM103 cells. The transformed cells are grown in LB medium supplemented with ampicillin (50 tzg ml-1). After 18 hr of cultivation at 37°, the cells are harvested by centrifugation. It is usual to obtain 40 g of wet cells from 10 liters of culture medium. Purification Purification of BCAT is carried out at 0-4 °. All buffers contain 2 mM 2-oxoglutarate, 0.2 mM EDTA, 5 mM 2-mercaptoethanol, and 0.1 mM 6 L. Clarke and J. Carbon, Methods Enzymol. 68, 396 (1979). 7 H. Ogawa and J. Tomizawa, J. Mol. Biol. 23, 265 (1967).
~ u
¢1
°
~i
~
106 ~
~
~.~
o0~
~,~
o~ ~
m ~
oo
o~a
.~:
o
~
o
~
I~
~,,.
o
o
o1~
~1
I~
[1 1] o
~
~
o
g
O.la
~ o.l.J
g ~
0
o
R
~,
oi~
o
u
I~1
o~
o~
o~
~
Itl
c~.u
ENZYME CLONING, EXPRESSION, AND PURIFICATION
o~
u
[11]
E. £oli BRANCHED-CHAINAMINOTRANSFERASE
107
pyridoxal 5'-phosphate (PLP). The cells (40 g) are resuspended in 100 ml of 0.1 M potassium phosphate buffer (KPi), pH 7.0, and are disrupted sonically at 0° for 10 min with a Branson (Danbury, CT) sonifier model 350 set at output 6 and 50% duty. Cell debris is removed by centrifugation (10,000g, 40 min). The supernatant is applied to a DEAE-Toyopearl 650M column (2.5 × 25 cm) equilibrated with 20 mM KPi, pH 7.0. The proteins are eluted with a l-liter linear gradient of 0 to 0.6 M NaCI in 20 mM KPi, and active fractions are combined. Solid ammonium sulfate is added to 20% saturation while maintaining the pH at 7.0 with 1 M K H 2 P O 4 . The enzyme solution is applied to a butyl-Toyopear1650M column (2.5 X 25 cm) equilibrated with 20 mM KPi, pH 7.0, containing 20% saturated ammonium sulfate. The proteins are eluted with a l-liter gradient of 20 to 0% saturated ammonium sulfate in 20 mM KPi, pH 7.0. The fractions containing BCAT are combined, and dialyzed against 4 liters of 20 mM KPi, pH 7.0, for 6 hr, and then against 4 liters of 2 mM KPi, pH 7.0, for 6 hr. The dialyzed solution is applied to a hydroxyapatite column (2.5 X 25 cm) equilibrated with 2 mM KPi, pH 7.0. BCAT is eluted with a l-liter linear gradient formed with 2 and 100 mM K P i , pH 7.0. The high-purity fractions are combined and concentrated to about 10 ml with an Amicon (Danvers, MA) ultrafiltration cell, and the concentrated solution is applied to a Sephacryl S-200 column (3.8 × 90 cm) equilibrated with 10 mM KPi, pH 7.0, containing 0.1 KCI. The enzyme is eluted with the same buffer. Generally, about 200 mg of BCAT is obtained from 40 g (wet weight) of E. coli JM103/pUCll9ilvE cells, with a yield of 40% and 20-fold purification. The final preparation of the enzyme has a specific activity of 0.4 kat kg -1. Crystallization and Crystallographic Analysis Crystallization of BCAT is performed in the dark from polyethylene glycol 400 (PEG 400) by vapor diffusion of a hanging drop. 8 The reservoir solution contains 100 mM HEPES (pH 7.5), 200 mM MgC12 as the salt, 30% (w/v) PEG 400 as the precipitant, 1 mM NAN3, 1raM EDTA, and 10 /zM PLP. Two different crystals, one belonging to the monoclinic space s K. Okada, K. Hirotsu, M. Sato, H. Hayashi, and H. Kagamiyama, J. Biochem. 121, 637 (1997).
FIG. 1. Nucleotide sequence of the ilvE gene and its neighborhood [S. Kuramitsu, T. Ogawa, H. Ogawa, and H. Kagamiyama, J. Biochem. 97, 993 (1985)]. The amino acid sequence deduced from the gene is shown in single-letter code. The DNA sequence is numbered from the beginning of the gene. A possible ribosome-binding site is underlined.
108
ENZYME CLONING, EXPRESSION, AND PURIFICATION
[ 111
group C2 and the other to the orthorhombic space group C2221, are obtained. An asymmetric unit cell contains three subunits of BCAT. Only the ethyl mercury thiosalicylate (EMTS) derivative of the monoclinic crystal is obtained as the heavy atom derivative. Therefore, selenomethionyl BCAT is prepared by expressing the BCAT protein in E. coli DL41 (metA-) cells harboring pUCll9-ilvE in the presence of selenomethionine. 9 Both forms of the crystal are obtained for the selenomethionyl enzyme. Data collection is performed at 2.4- or 2.5-A resolution. The monoclinic crystals of the native, the EMTS derivative, and the selenomethionyl enzymes are used for phase determination. Three mercury sites are located by using the isomorphous difference Patterson map calculated for the EMTS derivative. The positions of the 21 (7 × 3) selenium atoms are determined from difference Fourier maps phased with the mercury sites. It should be noted that the use of the selenomethionyl enzyme is not for applying the multiple anomalous dispersion method, as originally reported, 9 but for applying the conventional multiple isomorphous replacement (MIR) method. The initial MIR phase thus calculated is then improved by solvent flattening, 1° histogram matching, 11'12and noncrystallographic symmetry averagingJ 3 The improved electron density map is used for model building. Refinement is carried out with the X-PLOR program. The final R factor and free R factor are 18.8% for 37,577 reflections and 25.8% for 2044 reflections, respectively, with F > 2 [a(F)] between 10.0- and 2.5-A resolution, including 178 water molecules. Properties
Covalent and Noncovalent Structures The enzyme is composed of six identical subunits, each with Mr 33,960. 4'14'15The primary structure of the BCAT subunit is 26% homologous
to the subunit of the homodimeric enzyme D-amino-acid aminotransferase (DAAT) from Bacillus sp. YM-1 (Table I)J 6 Each subunit of BCAT, like
9 W. A. Hendrickson, J. R. Horton, and D. M. LeMaster, E M B O J. 9, 1665 (1990). 10B.-C. Wang, Methods Enzymol. 115, 90 (1985). 11 K. Y. J. Zhang and P. Main, Acta CrystaUogr. A46, 41 (1990). 12 K. Y. J. Zhang and P. Main, Acta Crystallogr. A46, 377 (1990). 13 G. Brigogne, Acta Crystallogr. A32, 832 (1976). 14F.-C. Lee-Peng, M. A. Hermodson, and G. B. Kohlhaw, J. Bacteriol. 139, 339 (1979). 15S. Kuramitsu, T. Ogawa, H. Ogawa, and H. Kagamiyama, J. Biochem. 97, 993 (1985). 16K. Tanizawa, S. Asano, Y. Masu, S. Kuramitsu, H. Kagamiyama, H. Tanaka, and K. Soda, J. Biol. Chem. 264, 2440 (1989).
[11]
E. coli BRANCHED-CHAIN AMINOTRANSFERASE
109
TABLE I KINETIC PARAMETERS OF BCAT TOWARD VARIOUS AMINO ACIDS AND 2-OxoGLUTARATEa k~tK~1 M -1 sec -1
Km]mM Substrate
keat/sec -1
Amino acid
2-Oxoglutarate
Amino acid
2-Oxoglutarate
Isoleucine Leucine Valine Methionine Phenylalanine Tyrosine Tryptophan
48 78 19 17 2.9 2.2 3.7
0.42 2.2 2.7 19 0.89 7.0 72
2.4 6.6 1.7 1.0 0.26 0.24 0.56
110,000 22,000 7,000 890 3,300 310 51
20,000 7,300 11,000 17,000 11,000 9,200 6,600
The reaction was carded out in 50 mM HEPES-NaOH, pH 8.0, containing 0.1 M KCI at 25°. The overall ping-pong reactions were followed by measuring the glutamate formed from 2-oxoglutarate. 4 The kcatK?~1 values toward 2-oxoglutarate are almost constant irrespective of the amino acid substrate, which is in accordance with theoretical considerations [H. Hayashi, K. Inoue, T. Nagata, S. Kuramitsu, and H. Kagamiyama, Biochemistry 32, 12229 (1993)].
DAAT, contains one molecule of pyridoxal 5'-phosphate (PLP). PLP is covalently bound to Lys-159 via an azomethine linkage with the e-amino group of the lysine side chain. 4 This and other active site residues, Arg-59 and Glu-193, later identified by X-ray crystallography, 8'17are also conserved in D A A T at the corresponding position (Fig. 2). BCAT and D A A T have no apparent sequence homologies with other aminotransferases. Among the PLP-dependent enzymes whose sequences have been determined, 4amino-4-deoxychorismate lyase, 18 encoded by pabC and catalyzing the last step of the biosynthesis of p-aminobenzoate (precursor of folate), is the only enzyme that is related in terms of primary structure to BCAT and D A A T (Fig. 2).
Spectroscopic Properties Because Lys-159 of BCAT and PLP form a Schiff base (PLP-Lys-159), BCAT has a major absorption band at 410 nm and a minor absorption band at 330 nm, similar to D A A T . 4'19 Both enzymes show negative circular 17 S. Sugio, G. A. Petsko, J. M. Manning, K. Soda, and D. Ringe, Biochemistry 34, 9661 (1995). xs p. V. Tran and B. P. Nichols, J. Bacteriol. 173, 3680 (1991). 19 K. Tanizawa, Y. Masu, S. Asano, H. Tanaka, and K. Soda, J. Biol. Chem. 264, 2445 (1989).
110 1 BCAT DAAT ADCM
i0
20
30
40
50
60
70
TTKKADYIWFNGEM-v'RWEDAKVHVMSHALHYGTSVFEGIRCYDSHKGPVVFRHREHMQRLHDSAKIYRFP ..... GYTLWNDQIVKDEEVKIDKEDRGYQFGDGVYEVVKVYN * * * * * * * * .......
FLINGHKQ *
.... ESLAVSDRATQFGDGCFTTARVIDG *
80
90
100
ii0
120
DAAT
I PYTKDKFHQLLHELVEKNELNTGHIYFQVTRGTSPRAHQFPENTVKPVI * ** * * * *
ADCM
I SCDFWPQLEQEMKTLAAE
BCAT
AMVS SWNRARPNT
DAAT
ATFVE-D*
150
RKNNLT
SAY I RPL I FVGDVGMGVNPPAGY
- QQNGVLKVVI
160
170
I PTAAKAGGNYL
IR-W-LRCDIKS* + - NPHLAG *
220
140
130
190 IALDVNGYI LHRNN *
*
*
-GIT
200
210
SEGAGENLFEVKDGVLFTP
- TVTEG S S SNVFG I KDG I LYTH ** * * *** * *
IKH - LNRLEQVL I RSHLEQTNADEALVLDSEGWVTECCAANLFWRKGNVVYTP + * * * * * * * *
230
240
250
ID
IGYTKE-NPRPLENLEKGVK *** * ** *
180
S S LLVG S EARRHGYQEG
*
S TDVI IAAF PWGAYLGAEALEQG
SRGSGGRGYSTLNSGPATRILSVTAYPAHYDRLRNE
LNLLGAVLAKQEAHEKGCYEAI * * * ** *
*
.... KVSLLSAHIQRLQDAC--QRLM * **
VSQ S I DELMEACRDVI
LAL S PVRLGR
.... GEMFTVNIDRLYASAEKIRIT * ** ** **
*
BCAT
ADCM
[11]
ENZYME CLONING, EXPRESSION, AND PURIFICATION
260
270
*
280
BCAT
PFTSSALPGITRDAIIKLAKELGIEVREQVLSRESLYLADEVFMSGTAAEITPVRSVDGIQVGEGRCGPV
DAAT
P~NNNILKGITRDWIAC~NEINNPVKEIPFTTHE.~&,KMDELFVTSTTSEITPVIEIDGKLIRDGKVGEW
ADCM
RLDQAGVNGIMRQFCIRLLAQSSYQLVEVQASLEESLQADEMVICN---ALMPVMPVC--A ** * * * **
290
**
..... CG-*
300
BCAT
TKR I QQAF FGLFTGETEDKWGWLDQVNQ
DAAT
TRKLQKQFETKIPKPLHI
ADCM
DVSF S SATLYEYLAPLCERPN
FIG. 2. The amino acid sequence of E. coli BCAT [S. Kuramitsu, T. Ogawa, H. Ogawa, and H. Kagamiyama, J. Biochem. 97, 993 (1985)], Bacillus sp. YM-1 D A A T [K. Tanizawa, S. Asano, Y. Masu, S. Kuramitsu, H. Kagamiyama, H. Tanaka, and K. Soda, J. Biol. Chem. 264, 2440 (1989)], and E. coli 4-amino-4-deoxychorismate lyase [ADCM; P. V. Tran and B. P. Nichols, J. Bacteriol. 173, 3680 (1991)]. The residues conserved between BCAT and D A A T are marked with asterisks below the sequence of DAAT. The residues conserved among the three enzymes are marked with asterisks below the sequence of ADCM. The PLPbinding lysine residues, identified in BCAT and DAAT, and proposed in ADCM, are marked with plus symbols.
dichroism (CD) bands at wavelengths corresponding to the absorption maxima. 4,19This is in contrast to most PLP-dependent enzymes, which show positive CD at the absorption maxima, 2° indicating the similar, unique properties of the two enzymes.
2o H. Hayashi, K. Inoue, T. Nagata, S. Kuramitsu, and H. Kagamiyama, Biochemistry 32, 12229 (1993).
[1 1 ]
E. coli BRANCHED-CHAINAMINOTRANSFERASE
111
Catalytic Properties
BCAT catalyzes transamination of a variety of amino acids (Table I). 4 Besides branched-chain amino acids, BCAT is active toward phenylalanine and tyrosine, both of which have bulky side chains like branched-chain amino acids. Methionine is also a fairly good substrate. While catalylzing transamination of these hydrophobic amino acids, BCAT is also active toward glutamate and its corresponding keto acid 2-0xoglutarate. Thus, like many other aminotransferases, BCAT recognizes two structurally different sets of substrates, amino/keto acids with hydrophobic side chains, and amino/keto acids with carboxylic side chains. Before the elucidation of the three-dimensional structures of BCAT and DAAT, an interesting stereochemical study had been carried o u t . E1 The key step in the catalytic reaction of aminotransferases is the 1,3-prototropic shift between the aldimine (Schiff base of an amino acid and PLP) and ketimine [Schiff base of a keto acid and pyridoxamine phosphate (PMP)] intermediates. For efficient removal of the a proton of the aldimine, the C~-H bond should be perpendicular to the plane of the PLP pyridine ring. Therefore, there are two possible conformations of the PLP-substrate aldimine: in one conformation the a hydrogen resides in the si face of the aldimine, and in the other conformation in the re face. The base that assists the prototropic shift is expected to locate on one side of the aldimine. Therefore, if the aldimine takes the conformation in which the a hydrogen protrudes to the si (re) face, the si (re) face base would abstract (and exchange with solvent) both the ct hydrogen of the aldimine and the proS (pro-R) hydrogen at C-4' of the ketimine. When apo-BCAT was reconstituted with (4' R)-[4'-aH]PMP and used as the catalyst for the transamination between 2-oxoglutarate and valine, the tritium was nearly completely released to the solvent. On the other hand, when (4' S)-[4'-aH]PMP was used for the reconstitution, essentially no radioactivity was detected in the solvent after the transamination reaction. Identical results were obtained for DAAT when it was used as a catalyst for the trasamination between D-alanine and 2-oxoglutarate. On the other hand, tritium was released only from (4' S)-[4'-3H]PMP in the catalytic reaction of AspAT (transamination between aspartate and 2-oxoglutarate), as expected from the crystallographic structure of AspAT. EETherefore, it was concluded that the base that catalyzes the 1,3-prototropic shift of the aldimine and ketimine is 21T. Yoshimura,T. Nishimura,J. Ito, N. Esaki, H. Kagamiyama,J. M. Manning,and K. Soda, J. Am. Chem. Soc. 115,3897 (1993). 22j. F. Kirsch G. Eichele, G. C. Ford, M. G. Vincent, J. N. Jansonius, H. Gehring, and P. Christen, J. Mol. Biol. 174~497 (1984).
112
ENZYMECLONING,EXPRESSION,ANDPURIFICATION
[11]
located at the re face of the PLP-substrate aldimine in BCAT and DAAT. The crystallographic structures of BCAT 8 and D A A T 17 are consistent with this conclusion. Crystallographic Structures
The hexameric structure of BCAT has 9 3 symmetry and the overall shape of a triangular prism (Fig. 3, left). The interaction between the subunits indicates that the hexamer can be regarded as a trimer of dimers. Each subunit is composed of two domains (Fig. 3, right). The larger domain is composed of residues 137-302. Residues 127-136, corresponding to the loop connecting the two domains, and the N-terminal three residues are missing in the crystallographic structure, probably because of their high flexibility in the substrate-unbound form of the enzyme. The orientation of the active site residues is similar to that of D A A T 17 (Fig. 4). Arg-59, which binds the phosphate group, and Glu-193, which interacts with the pyridine nitrogen of PLP, correspond to Arg-50 and Glu177, respectively, of DAAT. The side chain of Lys-159 locates at the re face of the PLP-Lys-159 aldimine. In DAAT, the PLP-Lys-145 Schiff base has the same conformation. This is in accordance with the notion that the
Trp126~ Gln137 .~_~
FIG. 3. T h e hexameric (left) and subunit (right) structures of B C A T (drawn using the coordinates of 1A3G). Left: Two sets of three subunits, related to each other by a noncrystallographic threefold axis, are separately drawn in gray (front) and black (back). Right: a - C a r b o n traces of one subunit. Residues 1 - 3 and 127-136 were not located on any electron density maps, and are not shown here. T h e P L P molecule is shown as a van der Waals surface model.
[11]
E. c o li BRANCHED-CHAINAMINOTRANSFERASE
AspAT
BCAT
113 DAAT
A
,~ %"Arg266
Tht257Gly256 59 ~ ~ _ _ _ Ala259
±s;%T ~5
~ _ . . ~ ! Ser243
B
+HNx\ //~-~, ~q
,~
_oy-
//INH+
O= o .-o
FIo. 4. (A) The active site structures of E. coli AspAT (left: 1ARS), E. coli BCAT (middle: 1A3G), and Bacillus sp. YM-1 DAAT (right 1DAA). (B) Schematicdrawing of the coenzyme-substrate amino acid Schiff base (aldimine). The orientation of the eoenzyme matches that in (A). In all structures, the a proton resides below the paper. The face below the paper is the si face of the aldimine in AspAT, and the re face of the aldimine in BCAT and DAAT.
e-amino group of Lys-159 of B C A T and Lys-145 of D A A T act as the reface base that catalyzes the 1,3-prototropic shift of the substrate-coenzyme Schiff base. The conformation of the substrate amino a c i d - P L P aldimine is schematically shown in Fig. 4B. In AspAT, Arg-386 is the residue that binds the a-carboxylate group of the substrate. The active site structure and the conformation of the amino a c i d - P L P aldimine of D A A T mirror those of AspAT, reflecting the enantiomeric specificity of these enzymes. Arg-98 and His-100 of D A A T are the proposed residues involved in the recognition of the substrate a-carboxylate group. While D A A T is active toward D-amino acids, B C A T recognizes L-amino acids. Therefore, compared with that in D A A T , the orientation of the substrate with respect to the coenzyme in B C A T is reversed. Thus, the a carboxylate is expected to point upward in Fig. 4. The amide nitrogens of Gly-256 and Thr-257 are the cadidates for the site that recognizes the a-carboxylate group of the substrate.
E. £oli BRANCHED-CHAINAMINOTRANSFERASE
103
now also been identified, 14-18 and we expect that reconstitution titrations with the catalytic subunits from the same organisms will become important tools in their biochemical characterization. We should also point out the usefulness of isolated A H A S subunits in the study of the physiology of branched-chain amino acid biosynthesis. Because of the tendency of at least some of the A H A S isozymes to dissociate on dilution, the intracellular activities of these enzymes may be underestimated when they are assayed. 7,19 We have used the addition of small subunits to the assay mixture to correct this p r o b l e m ) 9 If, as now seems likely, eukaryotic A H A S s also have a labile quaternary structure, the addition of the appropriate small subunits would also help clarify the real physiological activity of these AHASs.
Acknowledgment T h i s w o r k w a s s u p p o r t e d in p a r t b y G r a n t 243198 f r o m t h e I s r a e l S c i e n c e F o u n d a t i o n .
14N. Ohta, J. Plant Res. 110, 235 (1997). 15D. Chipman, Z. Barak, and J. V. Schloss, Biochim. Biophys. Acta 1385, 401 (1998). 16p. Bork, C. Ouzounis, C. Sander, M. Scharf, R. Schneider, and E. Sonnhammer, Protein Sci. 1, 1677 (1992). 17R. (3. Duggleby, Gene 190, 245 (1997). 18M. E. Reith and J. Munholland, Plant Mol. Biol. Rep. 13, 333 (1995). 19S. Epelbaum, R. A. LaRossa, T. K. VanDyk, T. Elkayam, D. M. Chipman, and Z. Barak, J. Bacteriol. 180, 4056 (1998).
[ 1 11 B r a n c h e d - C h a i n
Amino-Acid Escherichia
Aminotransferase
of
coli
B y HIROYUKI KAGAMIYAMA and HIDEYUKI HAYASHI
Ovemew In bacteria, branched-chain amino-acid aminotransferase (BCAT) catalyzes the last step of biosynthesis of branched-chain amino acids. Valine, leucine, and isoleucine are synthesized from the corresponding keto acids
METHODS IN ENZYMOLOGY, VOL. 324
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 0076-6879/00 $30.00
104
ENZYMECLONING,EXPRESSION,AND PURIFICATION
[1 1]
by amino-group transfer from glutamate. In Escherichia coli, B C A T is encoded by the gene ilvE, which is a component of the i l v G E D A operon. Earlier studies of E. coli transaminases indicated that there are two kinds of transaminase activities. Transaminase A is active toward dicarboxylic amino acids, and transaminase B is active toward branched-chain amino acids. 1 Transaminase A was later resolved into two enzymes: one has narrower specificity for dicarboxylic amino acids, and the other has broader specificity and catalyzes the transamination of aromatic amino acids as well as dicarboxylic amino acids. The first, high-specificity enzyme was identified as the E. coli counterpart of aspartate aminotransferase (AspAT), which is prevalent throughout species and is the most abundant and best-studied aminotransferase. 2,3 The second enzyme was determined to be an aromatic amino acid aminotransferase, and has an important role in catalyzing transamination between glutamate and aromatic keto acids, the last step in the biosynthesis of phenylalanine and tyrosine. 2'3 Transaminase B was clearly distinguished from these aminotransferases by the absence of activity toward aspartate. Structural bases for this difference have been provided both in primary and in three-dimensional structures, and are summarized in this chapter. A s s a y Method Principle
The enzymatic transamination between branched-chain amino acids and 2-oxoglutarate can be followed by measuring the production of glutamate through coupling to the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, using glutamate dehydrogenase, NAD, and 1-methoxy-5-methylphenazinium methosulfate. 4 In the original procedure, 5 diaphorase and 2-(p-iodophenyl)-3-(p-nitropheny)-5-phenyltetrazolium chloride were used in place of 1-methoxy-5-methylphenazinium methosulfate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide. Procedure
The assay mixture contains 50 m M H E P E S - N a O H (pH 8.0), 0.1 M KC1, 10 m M isoleucine, 10 m M 2-oxoglutarate, 2.5 m M N A D (oxidized 1D. Rudman and A. Meister, J. Biol. Chem. 200, 591 (1953). 2 D. H. Gelfand and R. A. Steinberg, J. Bacteriol. 130, 429 (1977). 3j. T. Powell and J. F. Morrison, Eur. Z Biochem. 87, 391 (1978). 4 K. Inoue, S. Kuramitsu, T. Ogawa, H. Ogawa, and H. Kagamiyama, J. Biochem. 104, 777 (1988). 5R. Rej, Anal. Biochem. 119, 205 (1982).
[i I]
E. coli BRANCHED-CHAINAMINOTRANSFERASE
105
form), 30 tzM 1-methoxy-5-methylphenazinium methosulfate, 0.2 mM 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, and 0.2 mg of glutamate dehydrogenase, in a total volume of 1.0 ml. The reaction is started by the addition of enzyme, and the absorbance at 500 nm is continuously monitored. The molar extinction coefficient is determined by measuring the absorbance change on addition of known amounts of glutamate to the assay mixture. The value is generally about 2 X 104 M -~ cm -a. Transamination between other amino acids and 2-oxoglutarate can be measured by substituting isoleucine with other amino acids. Cloning of ilvE Gene The chromosomal D N A of E. coli W3110 is prepared according to a standard method, 6 and digested partially with Sau3AI. The D N A fragments of about 1000 base pairs (bp) are collected on a sucrose density gradient] and ligated to the BamHI site of pBR322. The plasmid is used to transform E. coli AB2227 (ilvE-). The transformants that suppress the branchedchain amino acid auxotroph are selected on minimum agar plates supplemented with ampicillin (50/~g ml-1). The complete nucleotide sequence of the ilvE gene with its neighborhood and deduced amino acid sequence is shown in Fig. i. Purification Procedures The methods for overproduction and purification of BCAT are modified from the previously published methods. 4 Overproduction The cloned ilvE gene is excised at the SalI and X m n I sites flanking the gene, and is ligated to the SalI-SmaI site of pUCll9. The resultant plasmid, named pUCll9-ilvE, is used to transform E. coli JM103 cells. The transformed cells are grown in LB medium supplemented with ampicillin (50 tzg ml-1). After 18 hr of cultivation at 37°, the cells are harvested by centrifugation. It is usual to obtain 40 g of wet cells from 10 liters of culture medium. Purification Purification of BCAT is carried out at 0-4 °. All buffers contain 2 mM 2-oxoglutarate, 0.2 mM EDTA, 5 mM 2-mercaptoethanol, and 0.1 mM 6 L. Clarke and J. Carbon, Methods Enzymol. 68, 396 (1979). 7 H. Ogawa and J. Tomizawa, J. Mol. Biol. 23, 265 (1967).
~ u
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106 ~
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o~ ~
m ~
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.~:
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o
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~1
I~
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~
~
o
g
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ENZYME CLONING, EXPRESSION, AND PURIFICATION
o~
u
[11]
E. £oli BRANCHED-CHAINAMINOTRANSFERASE
107
pyridoxal 5'-phosphate (PLP). The cells (40 g) are resuspended in 100 ml of 0.1 M potassium phosphate buffer (KPi), pH 7.0, and are disrupted sonically at 0° for 10 min with a Branson (Danbury, CT) sonifier model 350 set at output 6 and 50% duty. Cell debris is removed by centrifugation (10,000g, 40 min). The supernatant is applied to a DEAE-Toyopearl 650M column (2.5 × 25 cm) equilibrated with 20 mM KPi, pH 7.0. The proteins are eluted with a l-liter linear gradient of 0 to 0.6 M NaCI in 20 mM KPi, and active fractions are combined. Solid ammonium sulfate is added to 20% saturation while maintaining the pH at 7.0 with 1 M K H 2 P O 4 . The enzyme solution is applied to a butyl-Toyopear1650M column (2.5 X 25 cm) equilibrated with 20 mM KPi, pH 7.0, containing 20% saturated ammonium sulfate. The proteins are eluted with a l-liter gradient of 20 to 0% saturated ammonium sulfate in 20 mM KPi, pH 7.0. The fractions containing BCAT are combined, and dialyzed against 4 liters of 20 mM KPi, pH 7.0, for 6 hr, and then against 4 liters of 2 mM KPi, pH 7.0, for 6 hr. The dialyzed solution is applied to a hydroxyapatite column (2.5 X 25 cm) equilibrated with 2 mM KPi, pH 7.0. BCAT is eluted with a l-liter linear gradient formed with 2 and 100 mM K P i , pH 7.0. The high-purity fractions are combined and concentrated to about 10 ml with an Amicon (Danvers, MA) ultrafiltration cell, and the concentrated solution is applied to a Sephacryl S-200 column (3.8 × 90 cm) equilibrated with 10 mM KPi, pH 7.0, containing 0.1 KCI. The enzyme is eluted with the same buffer. Generally, about 200 mg of BCAT is obtained from 40 g (wet weight) of E. coli JM103/pUCll9ilvE cells, with a yield of 40% and 20-fold purification. The final preparation of the enzyme has a specific activity of 0.4 kat kg -1. Crystallization and Crystallographic Analysis Crystallization of BCAT is performed in the dark from polyethylene glycol 400 (PEG 400) by vapor diffusion of a hanging drop. 8 The reservoir solution contains 100 mM HEPES (pH 7.5), 200 mM MgC12 as the salt, 30% (w/v) PEG 400 as the precipitant, 1 mM NAN3, 1raM EDTA, and 10 /zM PLP. Two different crystals, one belonging to the monoclinic space s K. Okada, K. Hirotsu, M. Sato, H. Hayashi, and H. Kagamiyama, J. Biochem. 121, 637 (1997).
FIG. 1. Nucleotide sequence of the ilvE gene and its neighborhood [S. Kuramitsu, T. Ogawa, H. Ogawa, and H. Kagamiyama, J. Biochem. 97, 993 (1985)]. The amino acid sequence deduced from the gene is shown in single-letter code. The DNA sequence is numbered from the beginning of the gene. A possible ribosome-binding site is underlined.
108
ENZYME CLONING, EXPRESSION, AND PURIFICATION
[ 111
group C2 and the other to the orthorhombic space group C2221, are obtained. An asymmetric unit cell contains three subunits of BCAT. Only the ethyl mercury thiosalicylate (EMTS) derivative of the monoclinic crystal is obtained as the heavy atom derivative. Therefore, selenomethionyl BCAT is prepared by expressing the BCAT protein in E. coli DL41 (metA-) cells harboring pUCll9-ilvE in the presence of selenomethionine. 9 Both forms of the crystal are obtained for the selenomethionyl enzyme. Data collection is performed at 2.4- or 2.5-A resolution. The monoclinic crystals of the native, the EMTS derivative, and the selenomethionyl enzymes are used for phase determination. Three mercury sites are located by using the isomorphous difference Patterson map calculated for the EMTS derivative. The positions of the 21 (7 × 3) selenium atoms are determined from difference Fourier maps phased with the mercury sites. It should be noted that the use of the selenomethionyl enzyme is not for applying the multiple anomalous dispersion method, as originally reported, 9 but for applying the conventional multiple isomorphous replacement (MIR) method. The initial MIR phase thus calculated is then improved by solvent flattening, 1° histogram matching, 11'12and noncrystallographic symmetry averagingJ 3 The improved electron density map is used for model building. Refinement is carried out with the X-PLOR program. The final R factor and free R factor are 18.8% for 37,577 reflections and 25.8% for 2044 reflections, respectively, with F > 2 [a(F)] between 10.0- and 2.5-A resolution, including 178 water molecules. Properties
Covalent and Noncovalent Structures The enzyme is composed of six identical subunits, each with Mr 33,960. 4'14'15The primary structure of the BCAT subunit is 26% homologous
to the subunit of the homodimeric enzyme D-amino-acid aminotransferase (DAAT) from Bacillus sp. YM-1 (Table I)J 6 Each subunit of BCAT, like
9 W. A. Hendrickson, J. R. Horton, and D. M. LeMaster, E M B O J. 9, 1665 (1990). 10B.-C. Wang, Methods Enzymol. 115, 90 (1985). 11 K. Y. J. Zhang and P. Main, Acta CrystaUogr. A46, 41 (1990). 12 K. Y. J. Zhang and P. Main, Acta Crystallogr. A46, 377 (1990). 13 G. Brigogne, Acta Crystallogr. A32, 832 (1976). 14F.-C. Lee-Peng, M. A. Hermodson, and G. B. Kohlhaw, J. Bacteriol. 139, 339 (1979). 15S. Kuramitsu, T. Ogawa, H. Ogawa, and H. Kagamiyama, J. Biochem. 97, 993 (1985). 16K. Tanizawa, S. Asano, Y. Masu, S. Kuramitsu, H. Kagamiyama, H. Tanaka, and K. Soda, J. Biol. Chem. 264, 2440 (1989).
[11]
E. coli BRANCHED-CHAIN AMINOTRANSFERASE
109
TABLE I KINETIC PARAMETERS OF BCAT TOWARD VARIOUS AMINO ACIDS AND 2-OxoGLUTARATEa k~tK~1 M -1 sec -1
Km]mM Substrate
keat/sec -1
Amino acid
2-Oxoglutarate
Amino acid
2-Oxoglutarate
Isoleucine Leucine Valine Methionine Phenylalanine Tyrosine Tryptophan
48 78 19 17 2.9 2.2 3.7
0.42 2.2 2.7 19 0.89 7.0 72
2.4 6.6 1.7 1.0 0.26 0.24 0.56
110,000 22,000 7,000 890 3,300 310 51
20,000 7,300 11,000 17,000 11,000 9,200 6,600
The reaction was carded out in 50 mM HEPES-NaOH, pH 8.0, containing 0.1 M KCI at 25°. The overall ping-pong reactions were followed by measuring the glutamate formed from 2-oxoglutarate. 4 The kcatK?~1 values toward 2-oxoglutarate are almost constant irrespective of the amino acid substrate, which is in accordance with theoretical considerations [H. Hayashi, K. Inoue, T. Nagata, S. Kuramitsu, and H. Kagamiyama, Biochemistry 32, 12229 (1993)].
DAAT, contains one molecule of pyridoxal 5'-phosphate (PLP). PLP is covalently bound to Lys-159 via an azomethine linkage with the e-amino group of the lysine side chain. 4 This and other active site residues, Arg-59 and Glu-193, later identified by X-ray crystallography, 8'17are also conserved in D A A T at the corresponding position (Fig. 2). BCAT and D A A T have no apparent sequence homologies with other aminotransferases. Among the PLP-dependent enzymes whose sequences have been determined, 4amino-4-deoxychorismate lyase, 18 encoded by pabC and catalyzing the last step of the biosynthesis of p-aminobenzoate (precursor of folate), is the only enzyme that is related in terms of primary structure to BCAT and D A A T (Fig. 2).
Spectroscopic Properties Because Lys-159 of BCAT and PLP form a Schiff base (PLP-Lys-159), BCAT has a major absorption band at 410 nm and a minor absorption band at 330 nm, similar to D A A T . 4'19 Both enzymes show negative circular 17 S. Sugio, G. A. Petsko, J. M. Manning, K. Soda, and D. Ringe, Biochemistry 34, 9661 (1995). xs p. V. Tran and B. P. Nichols, J. Bacteriol. 173, 3680 (1991). 19 K. Tanizawa, Y. Masu, S. Asano, H. Tanaka, and K. Soda, J. Biol. Chem. 264, 2445 (1989).
110 1 BCAT DAAT ADCM
i0
20
30
40
50
60
70
TTKKADYIWFNGEM-v'RWEDAKVHVMSHALHYGTSVFEGIRCYDSHKGPVVFRHREHMQRLHDSAKIYRFP ..... GYTLWNDQIVKDEEVKIDKEDRGYQFGDGVYEVVKVYN * * * * * * * * .......
FLINGHKQ *
.... ESLAVSDRATQFGDGCFTTARVIDG *
80
90
100
ii0
120
DAAT
I PYTKDKFHQLLHELVEKNELNTGHIYFQVTRGTSPRAHQFPENTVKPVI * ** * * * *
ADCM
I SCDFWPQLEQEMKTLAAE
BCAT
AMVS SWNRARPNT
DAAT
ATFVE-D*
150
RKNNLT
SAY I RPL I FVGDVGMGVNPPAGY
- QQNGVLKVVI
160
170
I PTAAKAGGNYL
IR-W-LRCDIKS* + - NPHLAG *
220
140
130
190 IALDVNGYI LHRNN *
*
*
-GIT
200
210
SEGAGENLFEVKDGVLFTP
- TVTEG S S SNVFG I KDG I LYTH ** * * *** * *
IKH - LNRLEQVL I RSHLEQTNADEALVLDSEGWVTECCAANLFWRKGNVVYTP + * * * * * * * *
230
240
250
ID
IGYTKE-NPRPLENLEKGVK *** * ** *
180
S S LLVG S EARRHGYQEG
*
S TDVI IAAF PWGAYLGAEALEQG
SRGSGGRGYSTLNSGPATRILSVTAYPAHYDRLRNE
LNLLGAVLAKQEAHEKGCYEAI * * * ** *
*
.... KVSLLSAHIQRLQDAC--QRLM * **
VSQ S I DELMEACRDVI
LAL S PVRLGR
.... GEMFTVNIDRLYASAEKIRIT * ** ** **
*
BCAT
ADCM
[11]
ENZYME CLONING, EXPRESSION, AND PURIFICATION
260
270
*
280
BCAT
PFTSSALPGITRDAIIKLAKELGIEVREQVLSRESLYLADEVFMSGTAAEITPVRSVDGIQVGEGRCGPV
DAAT
P~NNNILKGITRDWIAC~NEINNPVKEIPFTTHE.~&,KMDELFVTSTTSEITPVIEIDGKLIRDGKVGEW
ADCM
RLDQAGVNGIMRQFCIRLLAQSSYQLVEVQASLEESLQADEMVICN---ALMPVMPVC--A ** * * * **
290
**
..... CG-*
300
BCAT
TKR I QQAF FGLFTGETEDKWGWLDQVNQ
DAAT
TRKLQKQFETKIPKPLHI
ADCM
DVSF S SATLYEYLAPLCERPN
FIG. 2. The amino acid sequence of E. coli BCAT [S. Kuramitsu, T. Ogawa, H. Ogawa, and H. Kagamiyama, J. Biochem. 97, 993 (1985)], Bacillus sp. YM-1 D A A T [K. Tanizawa, S. Asano, Y. Masu, S. Kuramitsu, H. Kagamiyama, H. Tanaka, and K. Soda, J. Biol. Chem. 264, 2440 (1989)], and E. coli 4-amino-4-deoxychorismate lyase [ADCM; P. V. Tran and B. P. Nichols, J. Bacteriol. 173, 3680 (1991)]. The residues conserved between BCAT and D A A T are marked with asterisks below the sequence of DAAT. The residues conserved among the three enzymes are marked with asterisks below the sequence of ADCM. The PLPbinding lysine residues, identified in BCAT and DAAT, and proposed in ADCM, are marked with plus symbols.
dichroism (CD) bands at wavelengths corresponding to the absorption maxima. 4,19This is in contrast to most PLP-dependent enzymes, which show positive CD at the absorption maxima, 2° indicating the similar, unique properties of the two enzymes.
2o H. Hayashi, K. Inoue, T. Nagata, S. Kuramitsu, and H. Kagamiyama, Biochemistry 32, 12229 (1993).
[1 1 ]
E. coli BRANCHED-CHAINAMINOTRANSFERASE
111
Catalytic Properties
BCAT catalyzes transamination of a variety of amino acids (Table I). 4 Besides branched-chain amino acids, BCAT is active toward phenylalanine and tyrosine, both of which have bulky side chains like branched-chain amino acids. Methionine is also a fairly good substrate. While catalylzing transamination of these hydrophobic amino acids, BCAT is also active toward glutamate and its corresponding keto acid 2-0xoglutarate. Thus, like many other aminotransferases, BCAT recognizes two structurally different sets of substrates, amino/keto acids with hydrophobic side chains, and amino/keto acids with carboxylic side chains. Before the elucidation of the three-dimensional structures of BCAT and DAAT, an interesting stereochemical study had been carried o u t . E1 The key step in the catalytic reaction of aminotransferases is the 1,3-prototropic shift between the aldimine (Schiff base of an amino acid and PLP) and ketimine [Schiff base of a keto acid and pyridoxamine phosphate (PMP)] intermediates. For efficient removal of the a proton of the aldimine, the C~-H bond should be perpendicular to the plane of the PLP pyridine ring. Therefore, there are two possible conformations of the PLP-substrate aldimine: in one conformation the a hydrogen resides in the si face of the aldimine, and in the other conformation in the re face. The base that assists the prototropic shift is expected to locate on one side of the aldimine. Therefore, if the aldimine takes the conformation in which the a hydrogen protrudes to the si (re) face, the si (re) face base would abstract (and exchange with solvent) both the ct hydrogen of the aldimine and the proS (pro-R) hydrogen at C-4' of the ketimine. When apo-BCAT was reconstituted with (4' R)-[4'-aH]PMP and used as the catalyst for the transamination between 2-oxoglutarate and valine, the tritium was nearly completely released to the solvent. On the other hand, when (4' S)-[4'-aH]PMP was used for the reconstitution, essentially no radioactivity was detected in the solvent after the transamination reaction. Identical results were obtained for DAAT when it was used as a catalyst for the trasamination between D-alanine and 2-oxoglutarate. On the other hand, tritium was released only from (4' S)-[4'-3H]PMP in the catalytic reaction of AspAT (transamination between aspartate and 2-oxoglutarate), as expected from the crystallographic structure of AspAT. EETherefore, it was concluded that the base that catalyzes the 1,3-prototropic shift of the aldimine and ketimine is 21T. Yoshimura,T. Nishimura,J. Ito, N. Esaki, H. Kagamiyama,J. M. Manning,and K. Soda, J. Am. Chem. Soc. 115,3897 (1993). 22j. F. Kirsch G. Eichele, G. C. Ford, M. G. Vincent, J. N. Jansonius, H. Gehring, and P. Christen, J. Mol. Biol. 174~497 (1984).
112
ENZYMECLONING,EXPRESSION,ANDPURIFICATION
[11]
located at the re face of the PLP-substrate aldimine in BCAT and DAAT. The crystallographic structures of BCAT 8 and D A A T 17 are consistent with this conclusion. Crystallographic Structures
The hexameric structure of BCAT has 9 3 symmetry and the overall shape of a triangular prism (Fig. 3, left). The interaction between the subunits indicates that the hexamer can be regarded as a trimer of dimers. Each subunit is composed of two domains (Fig. 3, right). The larger domain is composed of residues 137-302. Residues 127-136, corresponding to the loop connecting the two domains, and the N-terminal three residues are missing in the crystallographic structure, probably because of their high flexibility in the substrate-unbound form of the enzyme. The orientation of the active site residues is similar to that of D A A T 17 (Fig. 4). Arg-59, which binds the phosphate group, and Glu-193, which interacts with the pyridine nitrogen of PLP, correspond to Arg-50 and Glu177, respectively, of DAAT. The side chain of Lys-159 locates at the re face of the PLP-Lys-159 aldimine. In DAAT, the PLP-Lys-145 Schiff base has the same conformation. This is in accordance with the notion that the
Trp126~ Gln137 .~_~
FIG. 3. T h e hexameric (left) and subunit (right) structures of B C A T (drawn using the coordinates of 1A3G). Left: Two sets of three subunits, related to each other by a noncrystallographic threefold axis, are separately drawn in gray (front) and black (back). Right: a - C a r b o n traces of one subunit. Residues 1 - 3 and 127-136 were not located on any electron density maps, and are not shown here. T h e P L P molecule is shown as a van der Waals surface model.
[11]
E. c o li BRANCHED-CHAINAMINOTRANSFERASE
AspAT
BCAT
113 DAAT
A
,~ %"Arg266
Tht257Gly256 59 ~ ~ _ _ _ Ala259
±s;%T ~5
~ _ . . ~ ! Ser243
B
+HNx\ //~-~, ~q
,~
_oy-
//INH+
O= o .-o
FIo. 4. (A) The active site structures of E. coli AspAT (left: 1ARS), E. coli BCAT (middle: 1A3G), and Bacillus sp. YM-1 DAAT (right 1DAA). (B) Schematicdrawing of the coenzyme-substrate amino acid Schiff base (aldimine). The orientation of the eoenzyme matches that in (A). In all structures, the a proton resides below the paper. The face below the paper is the si face of the aldimine in AspAT, and the re face of the aldimine in BCAT and DAAT.
e-amino group of Lys-159 of B C A T and Lys-145 of D A A T act as the reface base that catalyzes the 1,3-prototropic shift of the substrate-coenzyme Schiff base. The conformation of the substrate amino a c i d - P L P aldimine is schematically shown in Fig. 4B. In AspAT, Arg-386 is the residue that binds the a-carboxylate group of the substrate. The active site structure and the conformation of the amino a c i d - P L P aldimine of D A A T mirror those of AspAT, reflecting the enantiomeric specificity of these enzymes. Arg-98 and His-100 of D A A T are the proposed residues involved in the recognition of the substrate a-carboxylate group. While D A A T is active toward D-amino acids, B C A T recognizes L-amino acids. Therefore, compared with that in D A A T , the orientation of the substrate with respect to the coenzyme in B C A T is reversed. Thus, the a carboxylate is expected to point upward in Fig. 4. The amide nitrogens of Gly-256 and Thr-257 are the cadidates for the site that recognizes the a-carboxylate group of the substrate.