Gerze. 98 (1991) 141-145

141

Elsevier

GENE

03887

Nucleotide sequence of the LYS2 gene of Saccharomyces tyrocidine synthetase 1 (Recombinant

DNA;

Mary E. Morris* Department

lysine;

yeast;

a-aminoadipate

reductase;

tyrocidine;

cerevisiae:

antibiotic

homology

to Bacillus

brevis

synthesis)

and Sue Jinks-Robertson

of‘ Biology, Emory University. Atlanta, GA 30322 (U.S.A.)

Received by J.A. Gorman: 10 July 1990 Revised: 5 October 1990 Accepted: 10 October 1990

SUMMARY

The Saccharomwes cerevisiue L YS2 gene, which encodes a-aminoadipate reductase, an essential enzyme in the yeast lysine biosynthetic pathway, has been sequenced. A large open reading frame (ORF) has been identified which can specify a 1392-amino acid protein with a deduced M, of 155 344. A DNA database search using the translated L YS2 ORF as a probe has revealed significant aa sequence homology to the Bacillus brevis enzyme tyrocidine synthetase 1.

INTRODUCTION

Two different pathways for lysine biosynthesis have been described : the diaminopimelic acid pathway in bacteria and some lower eukaryotes and the a-aminoadipate pathway exclusively in lower eukaryotes (Umbarger, 1978). In the yeast Swchurotn~ces cerevisiue, lysine is synthesized via the z-aminoadipate pathway from a-ketoglutarate and acetyl CoA in eight enzyme-catalyzed steps; the first four steps occur in the mitochondrion and the remainder in the cytoplasm (reviewed by Bhattacharjee, 1985). To date, twelve complementation groups defining loci involved in lysine

Correspondence to: Dr. S. Jinks-Robertson, University,

1510 Clifton Rd., Atlanta,

Dept.

of Biology,

Emory

GA 30322 (U.S.A.)

Tel. (404) 727-63 12; Fax (404) 727-2880. * Present

address:

200 Longwood

Program

in Neuroscience,

Ave., Boston,

Harvard

Medical

School,

MA 02115 (U.S.A.)

Abbreviations: aa. amino acid(s); zAR, I-aminoadipate reductase; bp, base pair(s); kb, kilobase or 1000 bp; LYS2, gene encoding zAR; nt, nucleotide(s);

oligo,

oligodeoxyribonucleotide;

frame;

S., Saccharomyces; TY 1, tyrocidine

coding

TYl;

037X-I

ORF, synthetase

open 1;

reading

tyd, gene en-

wt, wild type.

I IY Yl:SO3

50 0

1991

Elsevier

Sc~rnce

Publishers

B.V.

(Biomedical

Diwkn)

biosynthesis have been identified genetically in yeast: LYSl, LYS2, LYSI-LYSlO and LYSlI-LYS16. The yeast LYS2 gene encodes the enzyme a-aminoadipate reductase (EAR) which, together with the product of the LYSS gene, converts the intermediate a-aminoadipate to x-aminoadipic-6-semialdehyde in a three-step reaction. In the first step, r-aminoadipate is activated by ATPdependent adenylation, a unique step among aa biosynthetic pathways. In the second step, the activated substrate is reduced by NADPH. The final step is the cleavage of the adenyl group from the reduced substrate to yield the product cc-aminoadipate-&semialdehyde. Available evidence indicates that the first two steps are catalyzed by the L YS2 gene product, while the LYS5 gene product catalyzes the final step (Sinha and Bhattacharjee, 1971). It has been suggested that L YS2 and L YS5 encode different subunits of the active 180-kDa xAR, which has been partially purified (Storts and Bhattacharjee, 1989). Not only is the multifunctional L YS2 gene product interesting from a biochemical perspective, but the LYS2 gene itself has also been exploited for genetic studies. Since the LYS2 gene encodes an essential enzyme in the lysine biosynthetic pathway, yeast strains with mutations in this gene require lysine for growth on synthetic media. Rever-

142 tants or strains which have acquired a wt gene by transformation can be selected by simply plating cells on synthetic medium lacking lysine. It is possible to select not only for the acquisition of a LYS2 gene by transformation or by

portion of the LYSZ gene using the shotgun procedure of Bankier and Barrel1 (1983). The complete nt sequence of the LYS'2region is presented in Fig. 2. The GenBank ac-

reversion of mutant lys2 strains, but it is also possible to directly select for lys2 auxotrophs arising as a result of spontaneous loss of LYS2-encoding plasmids or as a result of forward mutation of the wt gene. The ability to directly select &s2 mutants results from the capacity of S. cerevisiue strains lacking zAR activity to utilize z-aminoadipate as a sole nitrogen source (Chattoo et al., 1979). The only other yeast gene for which there is a comparable way to directly select for and against wt gene function is the URA3 gene

M36287.

(Boeke et al., 1984). The L YS2 gene has been cloned by complementation in a !r:s2 mutant yeast strain and has been used as a selectable marker in the construction of both replicating and integrative yeast transformation vectors (Eibel and Philippsen, 1983; Barnes and Thorner, 1986). The locus produces a 4.2-kb transcript, and the 5’ and 3’ ends ofthe mRNA have been mapped by primer extension and S 1 nuclease analysis, respectively (Eibel and Philippsen, 1983 ; Fleig et al., 1986). In addition, Fleig et al. (1986) sequenced approx. 1 kb of the 5 ’ end and 1.5 kb of the 3’ end of the gene and identified the putative N and C ends of an encoded ORF. Their analysis, however, left an approx. 3-kb gap in the LYS2 sequence. In this paper we report the complete sequence of the yeast LYS2 gene and the results of a DNA database search for proteins homologous to TAR.

EXPERIMENTAL

AND

DISCUSSION

(a) Sequencing of the LYS2 gene A map of the L YS2 region is presented in Fig. 1, with the 5’ and 3’ regions previously sequenced by Fleig et al. (1986) indicated. We sequenced both strands of the internal

t

cession

number

for the sequence

reported

in this paper is

(b) Predicted protein product Computer analyses of the L YS2 region were done using the program Genepro (Riverside Scientific Enterprises, Seattle, WA). The TACTAAC sequence characteristic of yeast introns (Langford et al., 1984) is not present in this region and thus, like most yeast genes, the LY.SZ gene appears to contain no introns. Translation of the nt sequence in all possible reading frames yielded a large ORF in only one of the frames (data not shown); the 5’ and 3’ ends of this ORF are identical to those identified previously by Fleig et al. (1986). The ORF specifies a 1392-aa protein with a predicted M, of 155 344; the aa composition of the predicted protein is similar to that of other eukaryotic proteins (data not shown). The codon usage of the large ORF was analyzed by comparing it to that compiled for 59 highly or 99 lowly expressed yeast genes (Pesole et al., 1988). The presumptive L YSZ-encoded ORF follows the codon usage pattern for lowly expressed yeast genes, which is in agreement with experimentally determined mRNA levels (Barnes and Thorner, 1986). The translated protein was examined for evidence of internal duplications using dot matrix analysis and none were evident (data not shown). The LYS2encoded protein would be expected to have an nt-binding domain since it activates its substrate by adenylation. A search of the sequence revealed no striking homology to the consensus sequence for kinase-type nt-binding sites (G/AXXXXGKS/T; Doolittle, 1986) or for the ATPbinding site of various aminoacyl tRNA synthetases (e.g., HIGH ; Schimmel, 1987). A region containing four leucine residues, each separated by 6 aa (residues 143, 150,157 and 164) may be important in heterodimer formation with the

i 1 kb

I

I

Fig. 1. Restriction map of the L YS2 region. The hatched box corresponds to the L YS2 ORF and the single line to surrounding chromosomal sequences. The blackened and open boxes below the map correspond to the regions sequenced by Fleig et al. (1986) and in this work, respectively. Only restriction sites for selected

6-bp cutters

are shown;

the positions

of the ones outside

of the region sequenced

in this laboratory

are from Fleig et al. (1986).

sequence

fragments

sheared

approx.

Microchemical nt sequence.

the corresponding

Facility)

were filled in by sequencing

of Staden

using the

The aa sequence

of the large ORF is below

oligos

(1986). Gaps in pDP4 (Fleig et al., 1986) using synthetic as primers.

plasmid

pDP6 (Fleig

analysis

X&z1 site

gel and inserting

from plasmid

clones were sequenced program

Random and aligned using the computer (Emory University

fragment

clones for sequence

from the upstream

1 kb in length on an agarose

the 4-kb BglII-PvuIl fragments

shearing

into the SmaI site of M13mplO. of Sanger et al. (t977) either strand

method

end-repaired

of the yeast LYS2 region. The sequence Hind111 site is given (see Fig. 1). Random by ultrasonically

downstream et al., 1986), size-selecting

were generated

to the second

Fig. 2. Nucleotide

z icr

144

* :=, . :=>

match across conservative

GWCDFVGCIHDIFQDNAEAFPERTCWETPTLNSDKSRSFTY~INRTSNIVAHYLIKTG VPYAQGKSIHQLFEEQAEAFPDRVAIVFE-------NRRLSYQELNRKLLEKG **..*...*****.* _* * . . *..** *.*.*

231-337 21-120

LYS2 tycA

all seqs. substitutions

IKRGDVVMIYSSRGVDLMVCVMGVLKAGATFSVIDPAYPPARQTIYL VQTDSIVGVMMEKSIENVlAILAVLKAGGAYVPIDIEYPRDRIQYIL , . . _. .. ***** . ** ** . . ** .

TGDLGRYLPNGDCECCGRADDQVKIRGFRIELGEIDTHISQHPLVRENIT TGDLAKWLTDGTIEFLGRIDHQVKIRGHRIELGEIESVLLAHEHITEAW **** _ * ** * ** * ****** *******.. . *

LYS2 tycA

*

GEIYVRAGGLILEGYRGLPELNKEKFVNNWFVEKDHWNYLDKDNGEPWRQFWLGP~RLYR GELCIGGVGLARGYWNRPDLTAEKFVDNPFV-----------------------PGEKMYR **. _ *** **. *.* ****.A xx

620-729 361-448

LYS2 tycA

*

*

* . .**

. * .

RTFLKKRLASYAMPSLIVVMDKLPLNPNGKVDKPKLQFPTPKQLNLVAENTVSETDDSQF RDYAAQKLPAYMLPSYFVKLDKMPLTPNDKIDRKAL--PEP---DLT~QSQ~YHPPR.* * .** * .**.** ** *.*. * * * .** *.

786-910 476-591

TNVEREVRDLWLSILPTKPASVSPDDSFFDLGGHSILATKMIFTLKKKLQVDLPLG'~IFK TETESILVSIWQNVLGIEKIGIR--DNFYSLGGDSIQAIQWARLHS-YQLKLETKDLLN * *. *** ** * ._ * *. * * * . .* .*

..

YPTIK YPTIE **** Fig. 3. Amino acid sequence and Sharp, positive

integers

in the original identity;

alignment

1989). Conservative between

0 and 25. Ser/Thr

DNA database

dots, conservative

of portions

aa replacements search

of the LYS2 and Iq’cA-encoded proteins. are those with a score greater

and Glu;Asp

(LOD > 104) correspond

was done using the program

to be conservative

to aa 303-332,618-647.687-716

matrix of Dayhoff

substitutions.

Four stretches

and 792-821

ofthe

CLUSTAL

(Higgins

(1978) resealed of aa similarity

LYSZ-encoded

protein.

to give detected

Asterisks,

substitution

gene product if xAR has the subunit gested by Storts and Bhattacharjee (1989).

f.YS.5

are also considered

Alignment

than 10 using the aa similarity

structure

sug-

(c) Similarity of the LYS2 ORF to tyrocidine synthetase 1 The GenBank DNA database (release 62) was searched using the translated LYS2 sequence as a probe to identify proteins with aa similarities (Henikoff and Wallace, 1988). In this search the probe was compared to each possible reading frame of every sequence in the database by aligning stretches of 30 aa. Ofthe similarities detected, the only ones with log-odds (LOD) scores greater than 104 were obtained in comparisons with the tyrocidine synthetase l-encoding gene (t~a.4) from Bacillus hrevis (Weckerman et al., 1988). The similarity at the aa level was clearly evident as a diagonal extending over approx. one-half of each protein in a standard dot matrix analysis (data not shown). A partial sequence alignmfient of the two proteins beginning at aa 23 1 of rAR and aa 21 of TYl is presented in Fig. 3. The two proteins are 28 y, identical over approx. 100 aa and then the sequences diverge over the next 300 aa. The similarity is clearly evident again beginning at aa 620 and 361 of the L Y$2- and tJ]cA-encoded proteins, respectively. There is 40”,, identity over a stretch of approx. 100 aa followed by short region of no similarity (57 aa in the LYS2-encoded protein and 28 aa in the t_vcA-encoded protein) and then another 100-aa stretch of 3 17, identity.

The obvious implication of the striking aa similarities between the yeast enzyme rAR and the bacterial enzyme TY 1 is that there is functional homology between the two seemingly unrelated proteins. TYl is one of three multifunctional enzymes involved in the synthesis of the cyclic decapeptide antibiotic tyrocidine (reviewed by Katz and Demain, 1977). It corresponds to the so-called light enzyme which initiates the nonribosomal synthesis of tyrocidine by activating, racemizing and becoming covalently linked to phenylalanine, the first aa in the peptide. This initiation step is somewhat analogous to the charging of tRNAs with their respective aa. As mentioned. srAR activates its substrate x-aminoadipate by adenylation and then reduces it; there is no evidence that the substrate becomes covalently attached to the enzyme. Given what is known about the two proteins, a conservative guess concerning possible functional homology is that the regions with aa similarity are important for substrate adenylation, although we note that the substrates are chemically quite different. Sitedirected mutagenesis of the homologous regions would be the first step in assessing the possible functional significance of these regions. In the case of the LYS2-encoded protein, the ability to directly select against wt gene function in vivo can be used in conjunction with the yeast transformation system to substitute defined mutations constructed in vitro for the wt gene. In addition, the negative selection

145 system for L YS2 allows one to readily isolate spontaneous loss-of-function mutations, thus making this gene ideal for structure-function studies.

Eibel, H. and Philippsen,

P.: Identification

LYS2 gene by an integrative Genet.

of the cloned

transformation

S. cerevisine

approach.

Mol. Gen.

191 (1983) 66-73.

Fleig, U.N., Pridmore, cartridges

R.D. and Philippsen,

for use in genetic

P.: Construction

of LYS2

of Saccharom.vces

manipulations

cerevi-

siae. Gene 46 (1986) 237-245.

Henikoff,

ACKNOWLEDGEMENTS

S. and Wallace,

nucleotide

We thank U. Fleig for generously providing the cloned LYS2 gene, Guy Benian for invaluable help with the Staden program and D.F. Steele for helpful comments on the manuscript. This work was supported by National Institutes of Health grant GM38464 and Emory University Research Committee funds to S.J.-R.

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