Analysis of the functional domains of biosynthetic threonine deaminase by comparison of the amino acid sequences of three wild-type alleles to the amino acid sequence of biodegradative threonine deaminase

245

Gene, 63 (1988) 245-252 Elsevier GEN 02319

Analysis of the functional domains of biosynthetic threonine deaminase by comparison of the amino acid sequences of three wild-type alleles to the amino acid sequence of biode~radative threonine deaminase (Recombinant DNA; genes, ilvA, ILVI, tdc; Escherikhia co& K-12; Salmonella typhimurium; Saccharomyces cerevisjae; isozymes; feedback inhibition; allosteric re~lation)

Bruce E. TailIon”*,

Robert Littleb and Robert P. LawtherP

n Department of Biology, University of South Caroba, Coi~mbia, SC 29208 (U.S.A.j Biogenetics Corp., Irvine, CA 92715 (U.S.A.) Tel. 714-851-7733

Tel. 8~3-777-6792, and b American

Received 1.5September 1987 Revised 6 November 1987 Accepted 17 November 1987 Received by publisher 29 December 1987

SUMMARY

The nucleotide sequence of the gene, ilvA, for bios~thetic threonine deaminase (Tda) from Salmonella typhimMri~mwas determined. The deduced amino acid sequence was compared with the deduced amino acid sequences of the biosynthetic Tda from Escherichiu coli K-12 (ilvA) and Saccharomyces cerevisiae (ILVl) and the biodegradative Tda from E. coli K-12 (tdc). The comparison indicated the presence of two types of blocks of homologous amino acids. The first type of homology is in the N-terminal portion of all four isozymes of Tda and probably indicates amino acids involved in catalysis. The second type of homology is found in the C-terminal portion of the three biosynthetic isozymes and presumably is involved in either (i) the binding or interaction of the allosteric effector isoleucine with the enzyme, or (ii) subunit interactions. The sites of amino acid changes of two E. coli K-12 ilvA alleles with altered response to isoleucine are consistent with the conclusion that the C-terminal portion of biosynthetic Tda is involved in allosteric regulation.

INTRODUCTION

Threonine deaminase (Tda) catalyzes the deamination of threonine to yield cl-ketobutyrate and ammonia. Two isozymes of Tda have been identiCorrespondenceto: Dr. R.P. Lawther, Department of Biology, University of South Carolina, Columbia, SC 29208 (U.S.A.) Tel. (803)777-6792. * Current address: Department of Biological Science, CarnegieMellon University, Pittsburgh, PA 15213 (U.S.A.) Tel. (412) 268-3179. 0378-l il9/88/%03SO

D 1988 Elsevier

Science Publishers

B.V. (Biomedicai

fied. One of these, biode~adative Tda, permits microorganisms to utilize threonine as a carbon source and it is allosterically activated by AMP (Shizuta and Hayashi, 1976). The structural gene, tdc, was recently isolated from E. coli K-12 (Goss and Datta, 1985) and the nucleotide sequence determined (Datta et al., 1987). The second isozyme, biosynthetic Tda, catalyzes the fast step in the Abbreviations: aa, amino acid(s); bp, base pair(s); Tda, threonine deaminase. Division)

246

synthesis of isoleucine. This enzyme has been studied from a variety of bacteria (Umbarger, 1956; Datta, 1966; Bums and Zarlengo, 1968; Hatfield and Umbarger, 1970), yeast (Zhnmerman et al., 1969; Betz et al., 1971; Katsunuma et al., 1971) and plants (Bryan, 1980). The nucleotide sequences of the ILVl gene of S. cerevisiue (Kielland-Brandt, 1984) and the i&A gene of E. coli K-12 (Lawther et al., 1987; Cox et al., 1987) have been determined. As initially described by Umbarger (1956), the biosynthetic isozyme is feedback-inhibited by isoleucine. Both biochemical and genetic techniques have been used to analyze isoleucine regulation of biosynthetic Tda. Treatment with mercurials of the biosynthetic Tda isolated from several bacterial species desensitizes the enzyme to inhibition by isoleucine (Changeux, 196 1; Freundlich and Umbarger, 1961; Datta, 1966; Maeba and Sanwai, 1966), while the enzyme from S. cerevisiae remains sensitive to isoleucine after treatment with mercurials (Betz et al., 197 1). A variety of genetic selection techniques have been used to isolate Tda variants with an altered response to inhibition by isoleucine. Feedback-resistant forms of Tda have been isolated by selection for resistance to thiaisoleucine (Betz et al., 1971) or glycyl-leucine (Vonder Haar and Umbarger, 1972) and by selection for the ability to use threonine as a nitrogen source in the presence of isoleucine (Bums et al., 1979). Tda hypersensitive to inhibition by isoleucine (Calhoun, 19’76)was isolated by selection for a variant of E. coli K-12 sensitive to inhibition by leucine (Levinthal et al., 1973). Determination of the nucleotide sequences of tdc and IL Vl allowed comparison of the deduced amino acid sequence of biodegradative Tda to the deduced amino acid sequence of biosynthetic Tda of S. cerevisiue. This analysis demonstrated several regions of sequence homology between these two proteins with 37% homology overall (Datta et al., 1987). To further understand the relationship of the structure of bios~thetic Tda to its catalytic and regulatory properties, the nucleotide sequence was determined for the wild-type i&A gene of S. typhimurium and two mutant alleles of the ilvA gene of E. coli K-12 (ilvA466 and ilvA538). Comparison of the deduced amino acid sequences of the biodegradative isozyme and the three wild-type

biosyn~etic alleles indicated the presence of several regions of homology. Regions of homology present in all four enzymes were located in the N-terminal portion of the enzymes while regions of homology exclusive to the three biosynthetic isozymes were found predominantly in the C-terminal portion of the enzymes. This distribution of sequence homologies may reflect a division of function between the N and C portions of these enzymes with the N-terminal portion being directly involved in catalysis and the C-terminal portion in feedback ambition. The sites of the ammo acid substitutions of the &A466 feedback-resistant mutation and the iZvA538 feedback-hypersensitive mutation are consistent with this proposal.

EXPERIMENTAL

AND DISCUSSION

(a) Nucleotide sequence of ihA ~y~~irnuri~rn

of ~&~rnoaeil~

The nucleotide sequence of the ilvA gene for biosynthetic Tda from S. typhimurium is presented in Fig. 1. Nucleotides that are different in E. coli K-12 are indicated beneath the sequence. Overall the nucleotide sequence is 85.5% homologous to that of ilvA from E. coli K-12. The majority of difTerences occur at nucleotides corresponding to the third base of an amino acid codon. fb) Comparison of the amino acid sequences of the isozymes of Tda The determination of functionally important segments of macromolecules by sequence comparison has been a highly useful technique. Comparison of the polypeptide sequence of the three biosynthetic isozymes to each other and to the biodegradative isozyme can indicate portions of the biosynthetic enzyme that are important in catalysis or in enzyme regulation. Fig. 2 presents a consensus analysis of the biosynthetic Tda from E. coli K-12 (Lawther et al., 1987 ; Cox et al., 1987), S. fyphimurium and S. cerevisiae (Kielland-Brandt et al., 1984) and biodegradative Tda from E. coZi K-12 (Datta et al., 1987). The amino acid sequences were aligned to maximize homology among the four isozymes using

241 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501

ATCGCGCAATCTCAACCTCTGTCAGTCGCGCCGGAAGGGGCGGAGTATCTGCGCGCGGTG T C G C CGT T T C A TAAA A CTACGCGCGCCAGTATACGAAGCGGCGCAGGTGACACCGCTGCAAAAAATGGAAAAGCTC G G T G T G A A G TCGTCACGTCTGGACAACGTTATTCTCGTCAAACGCGAAGACCGGCAGCCGGTACATAGC G 'T T C G G G T C A G C TTTAAGCTGCGCGGCGCTTACGCGATGATGACCGGATTGACCGAAGAACAAAAAGCCCAC A C GG CC G G G GGCGTGATTACCGCGTCGGCGGGGAACCATGCACAGGGCGTGGCGTTTTCCTCCGCGCGG C T T T T C G C T T CTTGGCGTGAAGTCGCTGATCGTCATGCCAAAAGCAACGGCGGATATTAAAGTCGATGCG TA CC T cc c c c c c C GTACGCGGTCTTGGCGGCGAAGTGCTGTTGCACGGCGCTAATTTTGATGAAGCGAAAGCG G CT C cc G C C AAAGCTATTGAACTGGCGCAGCAGCAGGGTTTTACTTGGGTGCCGCCGTTTGATCACCCA G C TA G C C C C T G ATGGTGATCGCCGGGCAGGGCACGCTGGCGCTGGAACTGCTTCAGCAGGATTCGCATCTC T A C CG C GATCGTGTCTTTGTGCCGGTTGGCGGCGGCGGCCTGGCGGCGGGCGTAGCGGTACTGATC C C A A C T T T G G AAACAATTGATGCCGCAAATCAAAGTGATTGCCGTCGAAGCGGAGGATTCCGCCTGCCTG C C A A C AAAGCGGCGTTGGAAGCCGGTCATCCAGTGGATCTCCCGCGCGTTGGGTTGTTTGCTGAA A C T G G T G A CA GGCGTAGCGGTAAAACGGATCGGCGACGAAACCTTCCGCCTGTGCCAGGAATATCTCGAC C T TT A G GACATTATTACAGTCGATAGCGACGCTATCTGCGCGCGGCGATGAAAGATCTGTTTGAAGAT c c c T G T G TA C GTGCGTGCGGTGGCCGAGCCGTCCGGCGCGCTGGCGCTGGCGGGGATGAAGAAATATATC C A C T A A GCCCAGCACAATATTCGCGGCGAACGACTGGCGCACGTCCTTTCCGGCGCCAACGTCAAC T C G TA T T G TTCCACGGCTTGCGCTATGTGTCTGAACGCTGCGAACTGGGGGAACAGCGCGAAGGCCTA C C C A C T CGT G CTGACGGTAACCATTCCGGAAGAAAAAGGGAACTTCCCCAAATTCTGCCAGTTGCTTGGC T G G CG T AC GGGCGTATGGTCACCGAATTTAACTACCGTTTTGCCGACGCGAAAAATGCCTGTATTTTT TC G C T C C c c CTCGGCGTACGCGTCAGTCAGGGGCTGGAGGAGCGAAAGAGATTATCACCCAACTGTGC TG CG C GC C C A C A T GCAGATG CAA GACGGCGGTTATAGCGTAGTGGATCTCTCCGACGATGAAATGGCGAAGCTGCATGTGCGC c c G T C A C TATATGGTCGGCGGACGCCCCTCCAAGCCATTACAGGAGCGTTTGTATAGTTTCGAATTT T A GCT G G A ccc c c C CCGGAGTCGCCCGGCGCGTTGCTCAAATTCCTGCATACGCTTGGCACGCACTGGAATATT A A G C GCGC CA C GT T C TCGCTATTCCATTACCGTAGCCACGGTACCGATTATGGCCGCGTGCTGGCGGCGTTCGAG TT G C T C T C C C G A A TTAGGCGACCATGAGCCGGATTTCGAAACCCGGCTGCATGAGCTGGGCTATGAATGCCAT CT A A C T C GATGAGAGCAATAACCCGGCGTTCCGGTTCTTTCTGGCGGGTTAA 1545 C AC A T G

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500

Fig.l. Thenucleotide sequence oftheilvA coding region ofS.typhimurium.The nucleotides that aredifferentinthe ilvA sequence of E.coli K-12areindicated beneath theS.typhimurium sequence. The nucleotide sequence was determined by thebase-specific chemical-cleavage method(MaxamandGilbert, 1980).

248

Cl I E.C.

s.t. S.C. g.c.

ilVA ilvA ILVl tdc

dntpDYvR1V . . . .mhityd 1pvAiddiia

LRapVYEaaq LRapVYEaaq LRsoVYDvIa akqrlagrIy

<

t

aadaqplaga 8aeaqplBva

lSlkLdelqt

pegAEY1RaV pegAEY1RaV

6

vtPLqkmekL

SSBtdnvILv

vtPLqkrakL .aPiaqgvgL

SSRLdavILv KREDrQPVhS SSRLntnViL KREDLlPVfS ScRckgaItL KfEnnQrtgS

kyg!lprrnyF

KREDrQPVhS

60

c2 E.C.

s.t. S.C. L.C.

ilvA ilVA ILVl tdc

1

1

PKLRGAY~Wf CKLRGAYallI FKLRGAYNWI

AgLTEeQKah tgLTEaQKah

AkLdDeQraq

GVItaSAGNM GVItaSAGNH GVIACSAGNH

FKiRGAFNkL

l sLTDaeKrk

GVVACSAGNH

AQGYAPSaAr AQGVAPSaAr AQGVAPaakh AQGVsLScAa

LGVkAlIVXP

taTadIKVdA

120

LGVkslIVHP kaTadIKVdA LkIpAtIVHP LGIdgkVVHP

vcTPsIKyqn LtgaPkaKVaA

c3 1 E.c. s.t. S.C. E.c.

ilVA ilvA ILVl

tdc

VrgFGgEVlL VrgLGgEVlL VarLGaqVVL tcdYaaEVVL

mVIAGQGTlA QVIAGQGTIA yVIAGQGTVA kVIAGQGTIg

1 LElLqQ.. .d LElCqQ.. .d HEILrQvrta LKIHed.. .l

KVIaVEeEDs KVIaVEaEDs

AcLkAaLdaG AcLkAaLeaG

hpvdLPrVG1 hpvdLPrVG1

KtIGVEtyDa rVIGVqsEnv

AtLhnSLqrn hgMeASFhsG

qrTpLPvVGT eiTthrttGT

KaiELsqqqG KaiELAqqqG ecakLAEerG Kv8EivEoeG

FTUVPPPDHP FTUVPPFDHP LTnIPPPDHP riFXPPYDdP

GGGGLaAGVA GGGGLaAGVA

VlIKqlaPqI VIIKqlsPqI

GGGGLIAGIg GGGGLIAGIA

aylKrVaPh1 VaIKsInPtI

HGaNPDEAKA HGaNFDEAKA yGndFDEAKA HGdNFnDtiA

177

C4 1

t E.C.

s. t. S.C. E.c.

IlvA ifvA ILVl tdc

ahlDrVFVPV shlDrVPVPV nkIgaVFVPV ydVDnViVP1

C5 ilvA s.t. ilvA S.C. ILVl E.c. tdc E.C.

GDETFRlcQE GDETFRlcQE GEETFRVaQq GalTYeIvrE

PAEGvgVkRI FAEGvaVkRI FADGtsVrmI LADGcdVsRp

ylDDIItVds ylDDIItVds vVDEVVLVnt 1VDDIVLVse

237

Rl DaICAAHKDL DaICAAMKDL DEICAAvKDi DEIrneMlaL

FEDvRaVaEP FEDvReVaEP FEDtRsIvEP iqrnkvVtEg

SGALAlAGMK SGALAlAGMK SGALsvAGHK aGALAcAaL1

297

TiPEekGsFl TiPEekGnFp TlPDvpGaCk

353

R2 E.C.

ilvA ilvA s.t. S.C. ILVl

KY1 . . . .alh KY1 . . , .aqh

E.o.

tdc

KYIstvhpEi 1Dq sgk....

E.C.

ilvA ilvA ILVl tdc

KfcqllgKRS KfcqllggRm KaqkiihpRS *.***.**.

nIrgerlah1 nIrgerlahV dhtkntyVp1 yIqnrktVsI

LSGANVNFhg LSGANVNFh~ LSGANmNFdR iSGgNIdLsR

LRYVSEReEL LRYVSERcEL LRFVSERavL vsqItgfvD8

GEqrEaLLaV GEqrEgLLtV GEgkEvFMlV *. . . . . . . .

. . . . . faD8k . . . . . faDak hrhesssEvp ...**..**

nACIFvgvr1 nACIFvgvrV kAYIYt8fsV ..*.****.*

srgleErKe1 SqgleErKeI vdrekEiKqV **..*.....

LQmLNdgGYs i tQLcdgGYs HQQLNalGFe .*.*.***..

405

ALlRFLn tLg ALlkFLhtLg ALtRFLggLs . . . . . . . . . .

tyWNi sLFHY thUNisLFHY

465

.

..........

d&m s.t. S.C. E.c.

l

VTEFnYR.. . VTEFnYR... VTEFsYRyne . . . . . . . ..I

l

RS

R4 E.c. s.t.

ilvA

E.c.

tdc

ilvA S.C. ILVl

vVDlSDdENA vVDlSDdENA aVDiSDnELA . . . . . . . . . .

KlHvRYHVGG KlHvRYMVGG KsHgRYLVGG . . . . . . . . . .

rpShplqER1 rpSKplqER1 .aSKvpnERi . . . . . . . . . .

ilvA s.t. ilvA S.C. ILVl E.c.

tdc

dsUN1 tLFHY . . . . . . . . . .

R7

RO E.C.

ySFEFPEsPG ySFEFPEsPG iSFtFPErPG . . . . . . . . . .

RsHGtDyGrV RsHGtDyGrV RnHGaDiGkV

LAa felgdhl? LA8fslgdhE LAgisvpprE

. . . . . . . . . .

. . . . . . . . . .

.pdFetrLnE .pdFe trLhE

LGYdCHDETn LGYeCHDEsn

NpaFrfFLag NpaFrfFL8g

nl tTqkfLeD LGYtYHDETd NtvYqkFLky . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* 515

* * .

Fig. 2. Comparison of the deduced amino acid sequences of biosynthetic Tda from E. coli K-t2, E. c. ilvA (Lawther et al., 1987; Cox et al.); 5’. fyphimurium, S. t. ilvA, and S. cerevisiae, S. c. ILVI (Kiellandt-Brandt et al., 1984), and biodegradative Tda from E. coli K-12, E. c. tdc (Datta et al., 1987). The comparison was accomplished using the PRETTY program (Devereux et al., 1984). Homologous amino

249

among subunits to form the active holotetramer. An E. coli B variant containing a nonsense mutation in the distal portionof the ilvA gene is consistent with this proposal (Feldner and Grimminger, 1976). The Tda isolated from this strain was neither inhibited by isoleucine nor did it bind isoleucine.

the PRETTY program of the University of Wisconsin Genetics Computer Group Sequence Analysis Software Package (Devereux et al., 1984) with the conserved amino acids being indicated by capital letters. The N-terminal sequence of ZLVI is truncated by 47 aa to correspond to the N terminus of ilvA. As described elsewhere, the N-terminal portion of IL VI is probably involved in the compartmentalization of Tda into the mitochondria of yeast (Kielland-Brandt et al., 1984). Five major regions of homology (designated Cl to C5) are present in all four Tda isozymes (Fig. 2). The first of these, Cl, corresponds to the Cl homology region described by Datta et al. (1987) for gene products of ZLV1, tdc and d&4 (D-serine deaminase from E. coli K-12). It has been proposed that this region participates directly in catalysis since the pyridoxal phosphate essential for enzyme activity is bound to the conserved lysine (aa 61 of ilvA) of tryptic peptides of both biodegradative Tda and D-serine deaminase (Datta et al., 1987). Moreover, Parsot (1986) noted that this is the only conserved lysine in the structure of threonine synthase from both E. coli and Bacillus subtilis, Tda from S. cerevisiae and D-serine deaminase from E. coli. It seems likely that the other four regions of homology between the four isozymes indicate domains involved in catalysis (i.e., catalytic amino acids, substrate binding site or amino acid residues required for the tertiary structure). A separate set of homologies exists only among the three biosynthetic isozymes (RI to R7). These homologies reside in the C-terminal portion of threonine deaminase. Since the biosynthetic enzymes are regulated differently from the biodegradative isozyme (i.e., feedback inhibition by isoleucine as opposed to activation by AMP), it seems likely that the amino acid sequences that are highly conserved among the biosynthetic isozymes are involved in regulation by isoleucine. These amino acid sequences would presumably be involved either directly in the binding of isoleucine or the interaction

acids are presented

in upper-case

was given a weight of 2 relative related open

compared boxes

biodegradative biosynthetic

Cl

Tda. The blackened Tda’s. The asterisks

To further investigate the segments of the biosynthetic Tda that participate in allosteric regulation, the nucleotide sequence of two mutant alleles of E. coli K-12 was determined. The ilvA466 mutation was isolated in strain CU5118 (Pledger and Umbarger, 1973a). The biosynthetic Tda in extracts from this strain was found to be resistant to inhibition by isoleucine (Pledger and Umbarger, 1973b). The subsequent purification of Tda from a derivative of this strain demonstrated that feedback resistance was the result of a change in the structure of Tda (Calhoun et al., 1974). The nucleotide sequence of the entire ilvA466 allele was determined and only a single bp difference from wild type was observed (Fig. 3A). The mutation is a C-to-T transition at bp 1441 (relative to Fig. 1) and changes leu-481 to phenylalanine (Fig. 3A). As indicated, this leucine is the fourth aa from segment R6. Analysis of purified Tda from a strain containing the ilvA466 allele indicated that the enzyme is resistant to feedback inhibition at pH 8.0, but is sensitive to inhibition at pH 6.0. Within 3 aa of the leucine-to-phenylalanine substitution is his-484. It seems likely that reducing the pH titrates this histidine, and possibly his-468, resulting in a change in the conformation of the protein sufficient for the enzyme to be inhibited by isoleucine. Isolation of the ilvA538 allele depended upon the observation that the growth of wild type E. coli K- 12 is temporarily inhibited upon the addition of leucine to minimal medium (Rogerson and Freundlich, 1970). The strain PS187 was isolated as an E. coli

indicate

boxes designated indicate

the protein

to maximize

homology.

The Tda sequence

This was done to allow for E. coli K-12 and S. fyphimurium

acids are numbered

to C5 (catalytic)

those

relative

to the deduced

sequences

Ri to R7 (regulatory) synthesis

and ilvA538

alleles

letters and gaps (...) have been introduced to the other sequences.

to S. cerevisiae. Amino

designated

(c) Sequence analysis of the ilvA466

terminating

among

indicate codon

of ilvA of E. cob K-12. The

amino acid sequence

conserved

the three

the sequences

of each respective

from S. cerevi.riue being very closely

biosynthetic

only conserved gene.

Tda’s among

and

the

the three

250

A R6 (11vA466)

T I

1396

CGCAGCCATGGCACCGACTACGGGCGCGTACTGGCGGCGTTCGAA~TTGGCGACCATGAA 1455 RsHGtDyGrVLAafelgdhe i

(4L)

B R2 (ilvA538) I CTGGCGCATATTCTTTCCGGTGCCAACGTGAACTTCCACGGC 969 T

928

1ahILSGANVNFhg

Fig. 3. Nucleotide

and amino acid sequence

ilvA466 allele was determined is numbered boxes,

relative

changes

from the plasmid

to the nucleotide

R2 and R6, are located

I

sequence

in the identical

of the iIvA466 and ilvA538 alleles of E. coli K-12. The nucleotide

pILVAFR.

The nucleotide

sequence

shown in Fig. 1. The amino positions

acid sequence

to the amino acid sequence

K-12 variant, the growth of which was completely inhibited by leucine (Levinthal et al., 1973). The leucine-sensitive phenotype of PS 187 depends, at least in part, upon the leucine sensitivity of Tda (~/VA 538) from this strain. Calhoun (1976) demonstrated that Tda from PS187 is hypersensitive to inhibition by isoleucine (i.e., Tda is inhibited at much lower concentrations of isoleucine than wild-type enzyme). The site of the bp change of the ilvA538 allele was ascertained (Fig. 3B). The altered properties of this enzyme result from the replacement of ala-3 17 by valine due to a C-to-T transition of bp 950 (relative to Fig. 1). As is indicated in Fig. 3B, ala-317 lies within homology region R2. As with the iZvA466 mutation it remains to be demonstrated whether these mutations affect subunit interactions or the binding of isoleucine.

of ilvA538 was determined is numbered

as presented

sequence

from pCATD538

of the and

as in Fig. 2. The blackened

in Fig. 2.

(d) Discussion Comparison of the deduced amino acid sequences of biosynthetic Tda of E. coli K-12, S. typhimurium, S. cerevisiae and biodegradative Tda indicate that two types of homologies exist among the enzymes. The first group (Cl to C5) of conserved amino acids is present in all four enzymes. The amino acids of this group are likely to be involved in the deamination of threonine. Consistent with this interpretation is that the presumptive pyridoxal phosphate binding site is among this group of homologies. The second set of homologies (Rl to R7) is found in the C-terminal portion of the three biosynthetic threonine deaminases. The amino acids of this group are presumably involved in either the binding of isoleucine or in subunit interactions. This interpretation

251

is consistent

with the deduced

amino

present in the Tda from two mutant

acid changes

alleles of ilvA of

also wish to thank assistance,

and

Donald

both

Mosser

Karen

for technical

Jackson

and

Debra

E. coli K- 12. In an effort to further identify the amino acids directly involved in the binding of isoleucine,

Williams for clerical assistance. This research was supported in part by grant GM28021 from the

the amino compared

National

acid sequence of biosynthetic to three other proteins that

leucine. This comparison which

catalyzes

synthesis

the final

(Kuramitsu

1987), isoleucyl

included:

Tda was bind iso-

transaminase

step in isoleucine

et al., 1985; Lawther

transfer

RNA synthetase

et al., 1984), and the isoleucine

binding

treatment

bio-

REFERENCES

et al.,

(Webster

protein pro-

of biosynthetic

of Health.

B

duct of the ilvJ gene (Landick and Oxender, 1985). Comparison of the deduced amino acid sequences of these proteins with Tda failed to identify any significant amino acid homologies. This indicates either that isoleucine-specific binding sites can be very different or that isoleucine binds in a unique way to each of these proteins. As described,

Institutes

Tda iso-

lated from E. coli K- 12 (Changeux, 196 1; Freundlich and Umbarger, 1961) or S. typhimurium (Maeba and Sanwal, 1966) with mercurials desensitizes the enzyme to isoleucine inhibition while the enzyme from 5’. cerevisiue (Betz et al., 1971) is unaffected. Since the overall secondary structure (using the Peptide Structure program; Devereux et al., 1984) of this enzyme from bacteria and yeast is very similar (not shown), comparison of the location of cysteines in the polypeptide chain could indicate the cysteine that is altered by mercurials. All of the cysteines found in Tda from E. coli K-12 are conserved in S. typhimurium (Fig. 2) and only cys-270 is also retained in the structure of Tda from yeast (Fig. 2). Thus, it is not possible to deduce which of the cysteines is altered by mercurials to desensitize Tda to inhibition by isoleucine. Threonine deaminase was the first enzyme reported to be feedback inhibited by the ultimate product of a biosynthetic pathway (Umbarger, 1956) and served as a model in the development of the concept of allosteric regulation (Changeux, 196 1; Monod et al., 1963). Continued study of this protein should lead to further insights into the relationship between primary structure and biological function.

Betz,

J.L.,

Hereford,

deaminase altered

in regulatory

Bryan,

Magee,

P.T.:

Threonine

cerevisiae mutationally

properties.

J.K.:

Synthesis

Biochemistry

of the aspartate

chain amino acids. Biochem. Bums,

R.O. and Zarlengo,

Plants

10 (1971)

M.H.:

family and branched5 (1980) 403-452.

Threonine

Salmonella typhimutium, I. Purification

deaminase

and properties.

from J. Biol.

Chem. 243 (1968) 178-185. Bums,

R.O.,

Hofler,

J.G.

and

Luginbuhl,

G.H.:

patterns

of inhibition

enzyme.

J. Biol. Chem. 254 (1979) 1074-1079.

Calhoun,

D.H.:

in an activator

Threonine

Calhoun,

enzyme

J. Bacterial. D.H.,

site-deficient

deaminase

feedback-hypersensitive mutant.

Escherichiu coli:

from

Kuska,

J.S. and

Hatfield,

G.W.:

properties

of the enzyme

lation of gene expression.

from a mutant

deaminase

altered

control mechanism

by L-isoleucine.

in its regu-

of biosynthetic

Cold Spring Harbor

Biol. 26 (1961) 313-318.

Cox, J.L., Cox, B.J., Fidanza, plete

Threonine and physical

J. Biol. Chem. 250 (1974) 127-131.

J.P.: The feedback

Symp. Quant.

regulatory

126 (1976) 56-63.

from Escherichia coli, II. Maturation

L-threonine

form of the

from a genetic

deaminase

Changeux,

Threonine

from Salmonella typhimurium. Substrate-specific

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and

1818-1824.

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