J. Biochem. 117, 276-282 (1995)

Three Conserved Glycine Residues in Valine Activation of Gramicidin S Synthetase 2 from Bacillus brevis1

Masaki Saito, Kazuko Hori, Toshitsugu Kurotsu, Masayuki Kanda, and Yoshitaka Saito Department of Biochemistry, Hyogo College of Medicine, Mukogawa-cho, Nishinomiya, Hyogo 663

Received for publication, July 27, 1994

The translated from the gene fragment containing the second and third domains of gramicidin S synthetase 2 was purified to an essentially homogeneous state. It showed valine and ornithine-activating activity and the second domain was proved to be the valine-activating domain. Three mutant genes from Bacillus brevis Nagano, BI-3, E-4, and E-5 strains, which encode defective valine-activating domains of gramicidin S synthetase 2, were sequenced. By comparison with the wild-type gene, single point mutations of guanine to adenine were found at the three conserved glycine codons; the 5303rd guanine in BI-3, the 5378th guanine in E-4, and the 4967th guanine in E-5, which corresponded to codon changes of the 1768th glycine to glutamic acid and the 1793rd and the 1656th glycine to aspartic acid. Loss of valine-adenylation activity by mutation at the 1656th glycine proved the direct participation of the TSGT/STGXPKG motif in the adenylation reaction, and suggests that this glycine residue with the conserved lysine residue of the motif forms the phosphate-binding loop for ATP-binding. The 1793rd glycine is a member of the YGXTE motif which was also conserved among adenylate-forming except acetyl-CoA synthetases. The 1768th glycine residue appears to maintain the conformation of the for aminoacyl adenylation since this residue is retained among the adenylate-forming enzymes, though flanking regions are not conserved. These results suggest that these glycine residues are essential for adenylate formation in the antibiotic peptide synthetase family and some other adenylate-forming enzymes.

Key words: aminoacyl adenylation, ATP-binding, Bacillus brevis, gramicidin S synthe tase 2, peptide synthetase.

A cyclic peptide antibiotic, gramicidin S (D-Phe L-Pro theine (8), and the two identical pentapeptides cyclize to L-Val-L-Orn-L-Leu)2 is produced nonribosomally by two form gramicidin S by an unknown mechanism. complementary multienzymes: gramicidin S synthetase 1 To clarify the reaction mechanism of such complicated (GS1) [EC 5.1.1.11] and 2 (GS2) (1). GS1, with a enzymes, we cloned and sequenced the entire nucleotide molecular weight of 120,000 activates, thioesterifies, and sequences of gramicidin S synthetase 1 and 2 genes (grsl racemizes phenylalanine, the first amino acid of gramicidin and grs2) (9-11). They were organized in an operon and S (2), and GS2 with a molecular weight of 510,000 acti had three open reading frames in the following order, grsT, vates and thioesterifies the other constituent amino acids, grsl, and grs2. The grsT gene encoded a protein of 256 proline, valine, ornithine, and leucine (3, 4). Individual amino acids homologous to fatty acid thioesterases. The amino acids are activated in a similar reaction to that grsl gene encoded a protein of 1,098 amino acids and the catalyzed by aminoacyl-tRNA synthetase (5), grs2 gene encoded a protein of 4,450 amino acids which consisted of four repeated domains of about 1,050 amino amino acid+ATP+Enz_??_aminoacyl AMP-Enz F PPi acids. The grsl protein and individual domains of the grs2 then the activated amino acids are transferred to protein had conserved 600-amino-acid sequences which sulfhydryl groups to form the aminoacyl-S-enzyme com were thought to be the activation sites for each of the plex (6), constituent amino acids of gramicidin S. Recently, several peptide synthetase genes have been cloned and sequenced aminoacyl AMP-Enz aminoacyl-S-Enz+AMP from bacteria and fungi (12-23). These proteins also had The synthesis of gramicidin S is started by a transfer of the conserved and repeated sequence of about 600 amino D-phenylalanine from GS1 to GS2 (7). The subsequent acids. The first, the third, and the fourth domains of grs2 polymerization process is followed by sequential thiolation protein were proved to be proline-, ornithine-, and leucine and transpeptidation reactions through 4•L-phosphopante activating domains, respectively (10, 11). In this paper, we report that the second domain of grs2 1 This work was supported in part by a research grant from Hyogo College of Medicine. protein is related to valine activation and that three Abbreviations: p-amidino PMSF, (p-amidinophenyl)methanesulfo conserved glycine residues of the valine-activating domain nyl fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel are essential for adenylate formation among aminoacyl and electrophoresis. acyl adenylate-forming enzymes.

276 J. Biochem. Aminoacyl Adenylation Site of Gramicidin S Synthetase 2 277

Fig. 1. Diagram of gramicidin S biosynthetic genes and construction of the expression vector containing the second and third domains of grs2 gene. The open box indicates the entire grs2 operon. pGS3KX, pGS3KX-S, and pGS2-7 were obtained by subcloning for sequencing (11). The recombinant plas mid pGS3KX19XX which expresses the second and third domains of grs2 gene, was constructed in pUC 19 by ligating the Xbal fragment of pGS2-7 with an insert of pGS3KX. Numerals under the box are nucleotide numbers of the grs2 gene from the deduced ATG initiation triplet.

were determined by direct sequencing of the genomic DNA. MATERIALS AND METHODS The chromosomal DNA preparations from the wild and mutant strains of B. brevis were obtained as described

Bacterial Strains and Plasmids•\A gramicidin S-pro previously (9) and used as templates. Two overlapped ducing strain of Bacillus brevis Nagano and its mutant fragments of grs2-val gene were amplified using two sets of stains, BI-3, E-4, and E-5 were used as sources of forward and reverse primers (24mers), 5•L-AGTACTTG chromosomal DNA (24, 25). Escherichia coli JM103(zPl) GGATTTCTCAGATCGG-3•L (primer 1; forward strand), [4(lac-pro), thi, strA, supE, endAsbcB, hsdR, F•LtraD36, 5•L-CTGTTGGGTAAGCATAATGCGTAC-3•L (primer 2; proAB, laclPZ4M15] and E. coli DH-1 [recA1, endAl, reverse strand), 5•L-TATCAGAAATCGAGATATTGTC gyrA96, thi-1, hsdRl7 (rk-, mk-), supE44] were used as TG-3•L (primer 3; forward strand), and 5•L-GCCCCTTC host cells. The plasmid vectors, pUC18 and pUC19, were AAATTCATAAAGGATG-3•L (primer 4; reverse strand). used for cloning or construction of recombinant plasmids. These primers annealed the nucleotides nt 2958 to nt 2981 The recombinant plasmids pGS3KX and pGS2-7 were (primer 1), nt 4810 to nt 4833 (primer 2), nt 4430 to nt obtained by subcloning for the sequencing of the grs2 gene 4453 (primer 3), and nt 6321 to 6344 (primer 4). The DNA as described previously (11). These inserts encoded a part sequences were determined by the dideoxy-nucleotide of the second domain and the full first or third domains of chain termination method of Sanger et al. (27). Sequenc the grs2 protein (Fig. 1). The recombinant plasmid, ing and analysis were carried out as described previously pGS3KX19XX, which expresses the second and the third (26). domains of the grs2 gene, was constructed in pUC19 by Biochemicals•\32PPi was purchased from the Radio ligating the pGS3KX insert with the smallest Xbal frag chemical Centre (Amersham, UK). Restriction endonucle ment of pGS2-7. The direction of the Xbal fragment in the ases were purchased from Takara Shuzo (Kyoto) and recombinant plasmid was confirmed by sequencing. Nippon Gene (Toyama). Gene AmpTM PCR reagent kit, Analysis and Purification of the Translated Product Ampli TagTM DNA polymerase, DNA ligation kit, and E. coli alkaline phosphatase were purchased from Takara from the Recombinant Plasmid pGS3KX19XX•\The trans lated product of the recombinant plasmid, pGS3KX19XX, Shuzo. DNase and RNase were obtained from Sigma was prepared on a large scale from the transformed E. coli Chemical (St. Louis, USA). All other chemicals were JM103LJP1 cells as described previously (9-11). An aliquot standard commercial products. of the ammonium sulfate fraction was separated by gel filtration FPLC on Superose 12 (Pharmacia) equilibrated RESULTS with buffer A [20mM sodium phosphate buffer (pH 7.5) containing 150mM NaCl, 1mM dithiothreitol, 1mM Identification of the Second Domain of Grs2 Gene as the Valine-Activating Domain The recombinant plasmid, MgC12i 0.5mM EDTA, 10 pM p-amidino PMSF, and 10% pGS3KX19XX, was constructed to express the full second glycerol] (11). Fraction numbers 24 to 28 from two-cycle and third domains of the grs2 protein (Fig. 1). The translat elution were pooled and applied to a Mono Q column ed product of pGS3KX19XX was partially purified by 50% equilibrated with buffer B [50mM Tris-HCl buffer (pH saturated ammonium sulfate fractionation from the lysate 8.0) containing 1mM dithiothreitol and 10% glycerol] and of the transformed cells as described previously (9, 10) and eluted with a linear gradient of 0 to 0.5 M NaCl in the same further purified by gel filtration FPLC on Superose 12 and buffer. Each fraction from Superose 12 and Mono Q column ion exchange FPLC on Mono Q. chromatography was used for assay of the amino acid The translated products of pGS3KX19XX had valine dependent ATP-32M exchange activities and for immuno and ornithine-dependent exchange activities. In the Super blot analysis. Immunoreactive proteins to GS2 antibodies ose 12 chromatography, these activities were eluted at the were analyzed by dot blotting and Western blotting as described previously (10, 26). Activities of the amino acid position corresponding to a protein of about 300kDa, which agreed with the deduced size of the expressed protein of the dependent ATP-32PP; exchange reaction were measured as insert. These fractions showed immunoreactivity to GS2 described previously (3). antibodies. DNA Sequence Analysis of the Mutant Grs2 Val Gene The Superose 12 fractions exhibiting the valine and the _??_m Various Strains of B. brevis-The sequences of the ornithine activation were subjected to FPLC Mono Q _??_ ne -activating domain from the wild and mutant genes

_??_ 17, No. 2, 1995 278 M. Saito et al.

Fig. 2. Purification of the translated product from pGS - 3KX19XX by ion-exchange FPLC on Mono Q. The active frac tions (numbers 24 to 28) obtained by gel filtration FPLC on Superose 12 were combined and subjected to FPLC on a Mono Q column equili brated with buffer B as described in "MATERIALS AND METHODS ." The column was eluted at a flow rate of 1ml/min with a linear gradient of 0 to 0.5M NaCl in the same buffer. Fractions of 0.5ml were collected. Amino acid-dependent ATP-32PPi exchange activities of 0.15ml of each fraction were mea sured. L-valine (•œ)-, L-ornithine

(•¢)-dependent ATP-32PPi ex change activities are shown. ---- protein concentration (A280); NaC1 concentration.

valine and ornithine-activating protein with a molecular mass of 300 kDa. As previously reported, the translated product of pGS3KX and pGS3KX-S exhibited only orni thine-dependent exchange activity (11) and these plasmids have only the third domain completely in the inserts, indicating that the third domain is the ornithine-activating site (Fig. 1). Therefore, the appearance of valine-activating activity in the product of pGS3KX19XX indicates that the second domain is the valine activation site. DNA Sequencing Analysis of Mutant Grs2 Val Gene To clarify the valine-activating mechanism of GS2, the second domain of grs2 genes (grs2-val) from BI-3, E-4, and E-5 strain of B. brevis was sequenced. These strains produce GS2 defective in valine activation (24, 25). The sequences were determined by direct sequencing using two overlapped fragments of grs2-val gene which were ampli fied by PCR. By comparison with the wild-type sequence Fig. 3. Western blot analysis and SDS-PAGE of the pGS3KX (11), the mutant grs2-val genes had single base changes of 19XX product. Aliquots of the lysate from the transformed cells, 0 guanine to adenine at nucleotide +5303 (BI-3), +5378 to 50% (NH4)2SO4 fraction of the lysate and the eluted fractions from (E-4), and +4967 (E-5), respectively. These nucleotide Mono Q chromatography were subjected to SDS-PAGE on 5-20% replacements result in the changes of the 1768th GGA polyacrylamide gradient gels, and proteins were transferred electro codon for glycine to the GAA codon for glutamic acid (BI-3), phoretically to a Durapore membrane and immunostained with the 1793rd GGC codon for glycine to the GAC codon for polyclonal antibodies to GS2, as described previously (10). Proteins were loaded as follows: lane 1, purified GS2 from B. brevis; lanes 2 aspartic acid (E-4), and the 1656th GGT codon for glycine and 3, lysates from E. coli JM1034P1 cells containing pUC19 (lane to the GAT codon for aspartic acid (E-5). 2) and pGS3KX19XX (lane 3), respectively; lanes 4 and 5, aliquots of Homology Search around the Mutated Residues of the fraction 75 from Mono Q chromatography, 0.8ƒÊl (lane 4) and 12ƒÊl Grs2 Val Proteins Defective in Valine Activation-Homol (lane 5). Lanes 1-4, Western blotting with anti GS2 antibodies; lane ogy search for the deduced amino acid sequence of grs2-val 5, SDS-PAGE by Coomassie Brilliant Blue staining. The sizes of molecular mass standards are indicated on the right. gene revealed that the 1656th and 1768th glycine residues are well conserved among adenylate forming enzymes, such as peptide synthetases, aminoacyl adenylating en column chromatography. Valine and ornithine-dependent zymes, and acyl adenylate-forming enzymes (10, 11). The ATP-32PP, exchange activities were eluted together, 1656-Gly is the first glycine residue of the TSGT/STGXP mainly at fraction number 75 (Fig. 2). Nearly homogeneous KG motif, which is also well conserved among adenylate forming enzymes and was suggested to be a phosphate protein with a molecular mass of 300kDa was seen in this fraction by SDS-PAGE with protein staining and a single binding loop in ATP binding (10). Therefore, the loss of immunoreactive protein to anti GS2 antibodies was seen at valine-activation activity by mutation at the 1656-Gly the same molecular mass by Western blotting (Fig. 3). residue indicates that this glycine residue is essential in the Immunoreactivites to GS2 antibodies also coincided with ATP-PP, exchange reaction and this motif is involved in the exchange activities on dot blotting (data not shown). adenylate formation. The 1793-Gly is a member of the These results indicate that pGS3KX19XX express YGXTE motif family, which is also conserved among

J. Biochem. Aminoacyl Adenylation Site of Gramicidin S Synthetase 2 279

Fig. 4. Comparison of the deduced amino acid sequence adja synthetase 1 (13); SRFA-1 to -7: the first to the seventh repeated cent to 1768-Gly and 1793-Gly of grs2-val domain with those of units in three large subunits of surfactin synthetase from B. subtilis adenylate-forming enzymes. A minimal number of gaps was (22); ACVS-A, -B, and -C: the first, the second, and the third repeated introduced to optimize the alignment; dark shadows indicate identical units in ACV synthetase from P. chrysogenum (15); HTS1-A, -B, -C, amino acids, and light shadows indicate chemically similar amino and -D: The first to the fourth repeated units of HC-toxin synthetase acids, defined as the following groups: A, S, T, P, and G; N, D, B, E, from C. carbonum (21); ENTF: E. coli EntF (28); ANGR: V. Q, and Z; H, R, and K; M, L, I, and V; F, Y, and W. The abbreviated anguillarum AngR (29); LYS2: S. cerevisiae LYS2 (30); LUC: firefly names to the left of the aligned sequences are as follows: GS2-VAL, luciferase (31); 4-CCL: parsley 4-coumarate: CoA (32); -PRO, -ORN, and -LEU: valine-, proline-, ornithine-, and leucine LCACS: rat long chain acyl-CoA synthetase (33); AAS: 2-acyl-GPE activating domains of B. brevis gramicidin S synthetase 2 (11); GS1: acyltransferase/acyl-ACP synthetase (34); ACS: acetyl-CoA synthe B. breuis gramicidin S synthetase 1 (9); TS1: B. brevis tyrocidine tase of A. nidulans (35). Asterisks indicate the mutated sites.

adenylate-forming enzymes except acetyl-CoA synthetases caused loss of valine-dependent ATP-"PP; exchange activ ity in mutant GS2 from BI-3, E-4, and E-5 strains of B. (Fig. 4). On the other hand, the flanking region of the 1768-Gly residue does not have significant similarity to any brevis. of the adenylate-forming enzymes, though 1768-Gly is The 1656th glycine residue is the first glycine residue of highly conserved among these enzymes (Fig. 4). Secondary the TSGT/STGXPKG motif, which is highly conserved and structure analysis by the methods of Chou and Fasman (36) thought to be a phosphate-loop for ATP binding in these showed that replacements of 1656-Gly or 1793-Gly residue adenylating enzymes (10, 15). The highly conserved lysine residue of this motif may be involved in the binding of the with an Asp residue did not result in any apparent change in the secondary structure, but replacement of 1768-Gly ATP based on the analogy of the lysine residue of the with a Glu residue results in the disappearance of ƒÀ-turn glycine-rich phosphate-loop GXXXXGKS/T, which is found in several nucleotide-binding proteins (37-39). In an structure at the mutated site and some changes in the X-ray crystallography study, Pai et al. reported that the secondary structure of the flanking region. This ƒÀ-turn equivalent lysine residue (Lys 16) in Ha-ras p21 protein, a structure is conserved among the adenylate-forming en GTP-binding protein, stretches across the phosphates of zymes. These results suggest that the 1656-Gly or 1793 GTP; its amino group is close enough to the ƒÁ-phosphate for Gly residue is functional in adenylate formation, whereas an ion-pair interaction and it forms a hydrogen bond with 1768-Gly may contribute to the maintenance of the three the main-chain oxygens of residues 10(Gly) and 11(Ala) dimensional structure. (38). These residues correspond to 1656-Gly and 1657-Thr in the valine-activating domain of the grs2 protein. The DISCUSSION same structural arrangement of lysine was also reported for the nucleotide-binding loop of porcine adenylate kinase, We have shown here that three conserved glycine residues an ATP-binding protein (39). Therefore, the loss of the in the valine activating domain, 1656-Gly, 1768-Gly, and exchange activity by the mutation of 1656-Gly to 1656-Asp 17 93-Gly, participate in the valine activation reaction. may be due to electrostatic hindrance by negatively charged Mutations of these residues to aspartic acid or glutamic acid

Vol.117, No. 2, 1995 280 M. Saito et al.

Fig. 5. Schematic diagram of the amino acid activating unit of grami cidin S synthetase 2. Shaded areas indicate the conserved region among the peptide synthetases and related adenyl ate-forming enzymes, with dark and light shading indicating relatively more or less homologous regions, respectively. Highly conserved motifs are shown as vertical solid bars. The boxed motifs and residue were proved to be involved in the adenylation reaction by our study. Asterisks and numerals of the boxed sequences or residue indicate mutation sites and amino acid numbers, as defined in this study, The numerals over the scale indicate the amino acid numbers of grs2 protein corresponding to the valine -activating domain.

aspartic acid of the interaction of the lysine residue with On the other hand, there is a relatively non-homologous phosphate of ATP. sequence of about 100-150 amino acids including the The 1793rd glycine residue is part of another highly conserved 1768-Gly residue, which is located between the conserved motif, YGXTE. This glycine residue exists p-loop sequence (TSGT/STGXPKG) and the recognition among all peptide synthetases and related carboxyl and binding sequence of the adenine moiety (QVKIRGX activating enzymes except acetyl-CoA synthetases. Acetyl RIE, TGD, and N/XGK). This non-homologous sequences CoA synthetases do not conserve this motif sequence may form the recognition space of the specific amino acid in strictly but have a slightly different sequence, Y/(W,F) individual domains as previously suggested (11). - WQTE. Since threonine and glutamic acid residues of this In peptide synthetases, the 600-amino-acid region ap motif are highly conserved among adenylate-forming pears to catalyze amino acid activation and formation of the enzymes and this motif forms a conserved ƒÀ-turn struc aminoacyl thioester. The carboxy-terminal region may be ture, these threonine and/or glutamic acid residues may be the site of the thioester formation, since this region con functional in the adenylation reaction, or for maintaining tains the LGGHSL motif, which is conserved only in the three-dimensional structure. Electrostatic repulsion of thioester-forming enzymes, such as acyl carrier proteins of 1796-Glu as a result of the substitution of 1793-Gly with fatty acid synthetase and polyketide synthetases, and is not negatively charged 1793-Asp may prevent the proper conserved in acyl adenylate-forming enzymes. These interaction of 1796-Glu or 1795-Thr with the . adenylating enzymes may have evolved from a common In addition to the above motifs and glycine residues, ancestral protein which catalyzed adenylation and thioester several other conserved motifs have been found in the formation using the thiol group of 4•L-phosphopantetheine. 600-amino-acid region (Fig. 5). We have previously report Although the amino acid activation -reaction in the ed that mutation at the 870-Gly residue of conserved peptide synthetases resembles that of aminoacyl-tRNA QVKIRGXRIE resulted in loss of proline-dependent ex synthetases, there is no sequence homology between the change activities and this glycine residue is essential for adenylation domains of the former and the latter proteins. aminoacyl adenylation (26). This motif occurs only in ami Probably they evolved separately from either class of noacyl adenylating enzymes but the corresponding glycine aminoacyl-tRNA synthetases. However, the adenylation residue is conserved even in acyl adenylating enzymes. domains of the peptide synthetases may form a nucleotide Recently Pavela-Vrancic et al. reported the contact site binding fold similar to the Rossmamn fold of ATP or GTP of the adenine moiety on ATP-binding by photoaffinity binding proteins (37, 41) and class 1 aminoacyl-tRNA labeling of TS1 with 2-azidoadenosine triphosphate (40). synthetases (42-44), involving the most conserved motifs Three sequences, 373-Gly-384-Lys, 405-Trp-416-Arg, such as SGTTGXPKG, YGXTE, QVKIRGXRIE, TGD, N/ and 483-Leu-494-Lys, have been identified. Two of them XGK, and several other conserved glycine residues in the correspond to 818-Gly-829-Lys and 850-Trp-861-Arg adenylating domain. near the 870-Gly residue of the proline-activating domain To clarify the adenylation mechanism of the peptide of GS2 (1853-Gly-1864-Lys and 1878-Arg-1885-Trp of synthetases more precisely, we are now studying the the valine-activating domain), and the TGD motif is found relationship between the conserved motifs and adenylation between the two sequences. The other labeled sequence reaction by introducing mutations at specific conserved 483-Leu-494-Lys, corresponds to 1978-Met-1985-Lys in sites. the valine-activating domain of GS2, and is flanked by the highly conserved motif N/XGK. These motifs are highly conserved among peptide synthetases and the TGD motif is REFERENCES considered as a putative ATP-binding region. 1. Tomino, S., Yamada, M., Itoh, H., and Kurahashi, K. (1967) There is another important motif, KAGGAY/F in the Cell-free synthesis of gramicidin S. Biochemistry 6, 2552-2560 adenylation domain of peptide synthetases. This motif is 2. Kanda, M., Hori, K., Kurotsu, T., Miura , S., Nozoe, A., and likely to be involved in amino acid recognition or binding, Saito, Y. (1978) Studies on gramicidin S synthetase: Purification since this sequence is not found in acyl adenylate-forming of the light enzyme obtained from some mutants of Bacillus enzymes. brevis. J. Biochem. 84, 435-441

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