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FEMS MicrobiologyLetters97 (1992)249-254 © 1992Federationof EuropeanMicrobiologica!S~:ietiest)3"/g-109"//q2/$t$.(g; Publishedby Elsevier 249 FEMSLE 05(195 Eight bacterial proteins, including UDP-N-acetylglucosamine acyltransferase (LpxA) and three other transferases of Escherichia coli, consist of a six-residue periodicity theme Martti Vaara De.partmem of Bactcriolo,gs"and Immtmoh)gy, Unirersity of tlelsb~ki, th.l.smki. Finland Received 17July 1992 Accepted 22 July 1992 Key words: Ipx.A Gene: ssc Gene: Lipid A biosynthesis: Hexapeptide repeat theme 1. SUMMARY Only a few prokauotie or eukaryotic enzymes are known to consist of a tandem-repeat structure. This report describes a common hexapept/de-repeat theme in four Escherichia colt transferases and in four less-characterized bacterial proteins The proteins are the Ssc protein of Salmonella typhimurium (25), UDP-N-acetylglucosamine acyltransferase of E. coli (24), the hypothetical proteins Tins of Bacillus subtilis (23) and Yglm of E. colt (22), succinyidiaminopimelate aminotransferase of E. colt (14), serine acetyltransferase of E. colt (13), NodL of Rhizobium leguminasorum (13), and thiogalactoside acetyltransferase of E. colt (8) (number of repeats indicated in parentheses). In UDP-N-acetyiglucosamine acyitransferase, the repeats constitute 55% of the total protein. Each hexapeptide re- Correspondence to: M. Vaara. Department of Bacteriology and Immunology,Universityof Helsinki.Haartmaninkatu3. 00290 Helsinki,Finland. peat of the eight proteins starts with lie, Lcu, or Val. Position b is occupied by Gly, position d by Gly, Asn, or Asp, and position e by Val or Ala in 52%, 54%, and 56% of the hexapeptide repeats, respectively. 2. INTRODUCTION Tripeptide GXY periodicity in collagens, the PGV and PGVGV periodicity in elastins, and the Gin-, Pro-, and/or Leu-rich oligopeptide repeats of cereal prolamines are well-known examples of the occurrence of tandem repeats in structural and storage proteins. Others include the imperfect a-helical heptapeptide repeats in coiled-coil proteins [I,2], the glycine-rich nonapeptide repeats in cytolytic RTX toxins of Gram-negative bacteria [3] and the flexible hinge sequences in immunoglobulins [4]. Zinc-finger and Lcu-zipper proteins as well as Ca2+-binding proteins with EF-hands contain functionally specialized domains with oligopeptide repeats. Repeats of longer oligopeptides (often longer than 10-15 residues) are a common feature in proteins involved in recognition processes. Enzymes of either prokaryotic or eukaryotic origin which are known to consist of a tandem repeat s,.ructure are extremely scarce. They include certain carbohydrate-binding streptococcal enzymes [5], the bacterial transfer RNA synthases with the coiled-coil structure [2], and RNA polymerase II. This study shows that five bacterial transferases, including an E. coli acyltransferase (UDP-N-acetylglucosamine acyltransferase; ref. 6), two E. coil acetyltransferases (thiogalactoside aeetyltransferase, serine acetyitransferase; refs. 7, 8), a probable acetyltransferase (the nodulation protein NOdL of Rhizobium leguminasorum; ref. 9), and an E. coli aminotransferase (succinyldiamino-pimelate aminotransferase; ref. 10), contain multiple successive imperfect-hexapeptiderepeat units. In UDP-N-acetylglucosamine acyltransferase, these hexapeptide repeats cover 55% of the total length of the protein. A large number of hexapeptidc repeats bearing this theme also occur in a recently discovered [11,12] enterobacterial protein Ssc, as well as in two hypothetical bacterial proteins. After this work had been completed, another report [13] described 26 hexapeptide repeats in the E. coli analogue of ssc. 3. MATERIALS AND METHODS FASTA searches [14] were made by using the University of Wisconsin GCG program package [15] (version 6.2) and the National Biomedical Research Foundation/Protein Identification Resource ( N B R F / P I R ) protein sequence database (release 26). When testing the theme (IGXXXX)61G, ktup 2 was used. The amino acid sequence of UDP-N-acetylglucosamine acyltransferase and the Ssc protein were deduced from GenBank entries M19334 [6] and M35193 [11], respectively. All protein homology alignments were made using the H1BIO PROSIS program package (Hitachi Software Engineering, Yokohama, Japan, ktup 1) and the SWISS-PROT database (release 14) included in this package. The secondary structures of the hexapeptide repeats were calculated by the method of Chou and Fasman [16] by determining the parameters P,, Po, P,, and p,. 4. RESULTS 4.1. Six.residue periodicity The Ssc protein of Salmonella typhimurium is a new bacterial protein, recently discovered and sequenced in this laboratory [11]. The sequence of its analogue in E. coli, the FirA protein, is also now known [17]; it is 96% identical to Ssc. The function of Ssc/FirA has not yet been elucidated but the protein has significant homology to E. coil UDP-N-aeetylglucosamine acyltransferase (the LpxA protein; ref. 18), which catalyzes the first fatty acid transfer step in the biosynthesis of the lipid A component of the outer membrane [19]. Furthermore, both Ssc and LpxA have been shown [19] to have sequence homology to the 'bacterial acetyltransferase family' [20,21] consisting of E. coil thiogalactoside acetyltransferase (LacA) and E. coil serine acetyltransferase (CysE), as well as of the NodL protein of R. leguminasorum. It was now realized that Ssc has regions notably rich in isoleucine residues and, more strikingly, that these residues were often regularly spaced. The distance between successive lies in Ssc was six residues in ten out of 33 occasions (Fig. la). Intriguingly, a similar six-residue periodicity was found in the isoleucine residues of NodL (Fig. lb). The isoleucine periodicity, although less pronounced, was also observed in LpxA, LacA, and CysE (data not shown). In those proteins, as well as in Ssc and NodL, the periodic lie was often followed by Gly, and was occasionally replaced by Leu or Val. An [I,V,L]G dipeptide residue was found altogether 54 times in these five proteins, (16, II, 11, 9, and 7 times in Ssc, LpxA, NodL, CysE, and LacA, respectively), and its occurrence conformed the six-residue periodicity pattern (Fig. lc). In only 26%, the distance between the successive [I,V,L]Gs was not proportional to six (10%, 27%, 30%, 33%, and 38%, for LpxA, Ssc, N o d e LacA, and CysE, respectively). 251 "ol t l o RESIDUE DISTANCE 10 011 1 • • RESIDUED~TANCE ,:tl 0.. I..li ,.... ~a c1 RES~(JE OI~TANCE Fig. I. (A) Periodic occurrence of lie residues in the Ssc protein of S. o'phimurium. The distance between each successive lie residue in Ssc was calculated. The figure shows that in ten occasions, the distance was six residues (i.e. lie residues were located in relative positions + I and + 7). (B) Isoleucine periodicity in the nodulation protein NOdLof R. leguminasorum. (C) Periodic occurrence of dipeptide residues IV, VG. and LG in S~, NodL, and the E. coil proteins thiogalactoside acetyltransferase (LacA), ~rine acetyltransferase (CysE), and UDP-N-acetylglucosamine acyltransferase (LpxA). The [i,V,L]-[I,V,L] distance between successive [IV,L]G residues in each of the five proteins were calculated, and the data combined. The hexad repeat arrangement of the five proteins is illustrated in Fig. 2a-b. Twenty-five more or less identical hexapeptide units, each starting with lie, Leu, or Val, were found in Ssc. Characteristically, t h e ~ units were clustered to form long stretches: they covered 44% of the total length of S ~ . The LpxA protein, aligned by PROSIS to reveal homology with Ssc, carried 24 analogous and similarly clustered hexads (covering 55% of LpxA). By identical criteria, NodL had 13, CysE 13, and LacA 8 hexad units. Other proteins which have been shown [19] to have homology with Ssc and LpxA include the hypothetical protein Yglm of E. coil [22] and its Bacillus subtilis analogue Tins [23]. Yglm is encoded by the flanking region of elmS (glucosamine phosphate isomerase) gene. Both Yglm and Tins carried 21 hexad units (Fig. 2c). Finally, limited homology has also been found [19] with succinyldiaminopimelate aminotransfcrase (DapD) of E. coil [10]. it had 14 hexads (Fig. 2d). The theme (1GXXXX),, used as (IGXXXX)61G, was then employed to screen all N B R F / P I R database proteins by the FASTA program. This theme was able to depict from all database proteins Ssc (10.0), NodL (10.0), LpxA (9.8), LacA (9.5), and Tins (8.2) as the best matches (values in parentheses indicate the deviation [in SDs] of the optimized score from the mean initial ,score of all database proteins). These were iollowed by bovine collagen a-2 (8.0). Altogether, 139 hexad units were found in the proteins shown in Fig. 2. 4.2. Amino acid occurrence in the hexads The occurrence of amino acid residues in different positions (a through f ) of the hexads is shown in Table 1. Besides the specificity of a and b, a clearcut feature was the predominance of Gly, Asn, and Asp in position d and that of Val and Ala in e. These findings were consistent for the hexads from each of the eight proteins (for details, see the footnotes of Table 1). Eighty-six percent (107/125) of the glycine residues occurred in b or d, and only 3% in positions e or f. Only 7% (8/110) of Val residues occurred in b, c, or d. Eighty percent (16/20) of Pro occupied c 252 A SSC: ~LPSIRLAD]J~OL~AZLHGD~D I V I TGVASMOSATTGHITFMVNPK¥ REHLGLCQASAV1/M'DODDLP FA~Sk,ALV1/KNPYLTYA~ I L D ~ P ~ L~XA: SSC: JIDATATI~ISNVS ~VIV~#rkNAV ~ E , , ~ I~DNVV .LGAGCF ~KNSK I~GSR ~ ~PSAV (105) M ~D KS~.F (?) ,IYHDI0.L~ENCL ~QSSTV,J~N)GFGYJ~ND~GNNVKI~LGRVI (20;*) ooo.o..,..,o.,,.o.o..,..,,o,,...,,o. $SC: ~RP'[TEgGVYS 5G1pLOPN1On~KTARLVHNZDDHSKRL~; = ' ~ O (341) UPXA: ~AOOV1PPWIAOG~HATPFG1/NIEGL~RRGFSRE&I~AI RNAYKLI YRSGKTLDZVKPE1A£ I J ~ T Y P E V I ~ F ~ F F ~ S T ~ L I R (262) B U~C&: NCX)L: C'~5E: MNY~HTERI P,N3K LF~DRCEGLPEKRLRGKT~*J~ m" R-~KZ ~ML&GE/4YNArDPEI OREL L LTGANLK HSCEEL£IVI~NNI KA£ARTIJ~DCEPHLASFYH/~TLLKHENLGSAL5Y,v~/~NKLSSPIMPAI AZ R£VVEEAYAADPE M I A S A A C O Z Q & ~ P A ~ K ¥ S (34) 434) (100) 1J~CA: EFNHSHPSEV~KRESLI KEMFAT ~ E I ~ N V£ PPWFS TGSNIH VGRN~y ANFNLT IVDDYW'~ Z~GDNVL~JUPN'VT13I'SV"/GHPVHHELRKNGEH ¥SFPIT (133) NODL: R1~)TLGDSAERNNGLL ~ERLGA~LGAV ~R PPFHCDYGFNIR~I~AWVY~N~'NCV~LDVAAVTI~DGTA~PAVO ~¥TADHPHDPEQROAGLQ ~RPVS (135) CYSE: TPLLYLKGFHJ~,OAYR IGHNLI~1~GRRRL~ IF ~NOV$ VTFQVD ~HPAAK ~IRGIH •I*Df~TGIV ~ £ T A V .~£NDVS 11I~OSV~mLGGTGKSGCORHPK 4195) IJ~C~: ~.~k~VW ~ 5 h ' V l ~NI~'I~ ~.~DNSV J ~ G S I ~KDIPPNVVAAGVPCRVIREINDRDKHYYFRDYKVESSV (203) NODL: I~GRHAN~.~GGAI ~LPGVT J..GDI~V I~A!35V mVTRDVpAGSTM4GNPARVK~GGRLPKS (ZgO) C~5£: (2?3) ~R£~ ~k~/kCd~K~LG~IE ~RG/kK ~m~/~SV ~q.OPVPPH~AAGVPARIVGKPDSDKPSMDHDOHFNGINHTF£¥GDG! C ~: TGLM: P~)KRFA~P.m(SKLYKV[JiPVCGKPHVEHV VOE/tIJ( LSLSKL MLNN/kHSWIIJ~GKG~HYSDLPKVLHTLJEGKAHV~HV ~O~NE ~ & J ~ V (49J (51) TMS: VTIVG~.,&EEVKKQ-LGOKSE¥ RVQN<Q~TJ~A ~/KQ~PFI.~DEKGVTIVICGDTPLLTAETt~QMLKEHTQREN~RTILTAVABDPTGyGRIIRSENG&V~KIV TGI21: HLVYGHGGDLLMQALI~)DNLNW VLOAEQ~TGHA~AAPFFADDEDI-IJILYGDVPLISVETL-QRLRD-AKPOGGIGLLTVKLDDPTGYGRITR-ENGKVTGZV (lb4) (153) T~tS: EHKDASEEERLVTEINTGTYCFDNEALFRAIDQVS~NAQGEYYLPDVIEZLKNEGETVAAyQTGNFOET ~VNDR VALSOAEOFMKERINXRI~NGVTLIDPHNTY (262) YGM4: b~tKDJLTDEO~I(~EINTGILIANG~MKPddIJ~;VTN~QGEY¥ITDII/~L~yQ£GREIVAVHPQRLSEV EGVNNR LQLSRLERVYQ$EQAEKLLLAG'~ILRDPARFD "n,~s: .l.sPo^v .L~sD'T'{ 2.~rPG'rv .;.KG~VQ ]GEDTZ ..~P,TE ;raSP, ZC~niV ;KQ-S mVVN,SK ',~NDW ZGPrP,H .;SeDSV ;GNEVK ]..GNrVt ;KK'tQrG • • ,,*,,,,,.,,,,o. lttS: DP.SK~HLSWGDAJE ~ V N ,o,,,oo, ........ am • mm Im ms • (261) O~S) ,o,, ~j~CGSTCVNYDGKNKYLTK;EDG&F T_GGCNSNLV/~PV~ ~EGAY _VNtGST BVTEDVPGKN~IAR/~QVNKDDYVKNIHK~ lq3I~q: ~GSK~HLT~'D~E ~.~DNI~ ~.~&GI'I~CN~DG~NKFK'~ ~'DDVF ~$DCO I~tVh.PV'i"~KGkT ~AJ~TF mV33NVGENAIJ~ISRVPOl~KEGNRRPVKKX (456) (456) D DAPD: H~t~N~TAFERR~E~TPANN7Fv~R~DN~V~AL~J)5G~kLRVA£K~DC~NV~H~LKK~VLL~FRINDN~V1~GA£~R~V~D~K~GVR~P~V~ ,,,,,,,,,,*,o,.o..,.,,,..,,,,, ~J~oD: ~YI)RETG¢IHYGm/PJU3SVVVSGNLPSKDGX¥SLYCAVIVKKVD~KTRGK ~INEL ~RTID (114) ,,.,,., (274) 253 Table I Percentage occurrence of amino acid residues in each position of the hexapeptide ~ Residue Position Tip a 58.3 b 25.2 16.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Cys 0.0 lie Val Leu Met Gly Ala Ser Thr Pro Ash Asp Gin Glu Arg Lys His Phe Tyr b c d e f 0.7 3,6 5.0 0.7 51.8 0 7.2 2.2 1.4 0.0 3.6 6.5 1.4 5.8 2.9 1.4 2.2 0.0 2.2 0.7 2.2 0.7 0.7 ! .4 10.1 15.1 7.9 4.3 I 1.5 8.6 12.2 2.9 10.1 4.3 2.9 2.9 0.7 0.7 0.0 0.0 1.4 0.7 1.4 25.2 c 6.5 4.3 2.2 2.9 18.7 c I0.1 c 2.2 1.4 5.0 0.7 7.9 5.0 0.7 1.4 2.2 32.4 c 0.0 0.7 1.4 23.7 c 10.8 12.2 (1.0 2.2 1.4 0.0 1.4 0.0 0.0 2.2 0.0 0.0 0.0 9.4 15.8 2.9 4.3 1.4 5.0 5.0 13.7 0.0 3.6 0.7 5.8 8.6 4.3 5.8 3.6 3.6 4.3 2.2 2.2 9.4 0.7 0.7 0.0 a The tandem hexapeptide repeats (n = 139, shown in Fig. 2 and filling the criteria in the legend of Fig. 2) were included in the analysis. b 72%, (Ssc); 46%, (LpxA); 88%, (LacA): 62%, (CysE): 77% (NodL); 43% (Yglm); 62% (Tins); 36% (DapD). c Val+Ala: 48%, (Ssc); 54%, (LpxA); 76%, (LacA); 69%, (CysEP.69% (NOdL); 57% (Yglm); 48% (Tmsk 50% (DapD). d 52%, (Ssc); 42%, (LpxA); 75%, (LacA); 38%, (CysE); 69% (NodL); 52% (Yglm); 62% (Tms); 36% (DapD). = Gly+Asn+Asp: 56%, (Ssc): 29%, (LpxA): 88%, (LacA): 54%, (CysE); 46% (NodL); 62% (Yglm); 57% (Tmsk 64% (DapD). a n d 72% ( 1 3 / 1 8 ) o f Cys in e. T h e majority o f A s n (75%, 3 8 / 5 1 ) a n d A s p (72%, 3 1 / 4 3 ) w e r e in cord. T h e m e t h o d o f C h o u a n d F a s m a n [16] p r e dicted a major p a r t o f the hexads to contain a //-turn. This was e x p e c t e d on the basis o f the a b u n d a n c y o f residues with h i g h - b e n d potential (such as Gly). Forty-nine p e r c e n t ( 6 8 / 1 3 9 ) o f the t e t r a p e p t i d e s bcde (i.e. p e p t i d e s with s u b s e q u e n t residues b, c, d, and e o f t h e hexad) had p, > 0.75 × 10 -4 (a value indicating a p r o b a b l e b e n d [at b]) as ",veil as ( P , ) > 1.00 a n d ( P , , ) < (P~) > <P0)" F o r the bcde p e p t i d e s from individual p r o teins, t h e c o r r e s p o n d i n g p e r c e n t a g e was: 75% (LacA), 62% (Tins), 50% ( D a p D ) , 50% (LpxA), 48% (Ssc), 48% (Yglm), 31% (CysE), and 31% (NodL). 5. D I S C U S S I O N T h e p r e s e n t finding indicates that e n z y m e s such as t r a n s f e r a s e s can b e m a d e from hexad blocks. This might h e l p b o t h in u n d e r s t a n d i n g m o l e c u l a r evolution o f e n z y m e s a n d in p l a n n i n g genetically e n g i n e e r e d enzymes. It would b e interesting to evaluate w h e t h e r o t h e r e n z y m e s show any signs o f periodicity (with a d i f f e r e n t pattern). LpxA, LacA, a n d D a p D are all E. coil transferases but have very d i f f e r e n t t r a n s f e r a s e functions. L p x A transfers an (R)-3-hydroxymyristoyl moiety from (R)-3-hydroxymyristoyl-acyl carrier p r o t e i n to the 3 - O H o f U D P - G I c N a c and has e x t r e m e specificity for acyl-chain length and an (R)-3-hydroxy function [18,24]. L a c A transfers the acetyl g r o u p o f acetyl-coenzyme A to t h e 6 - O H o f thiogalactosides a n d certain galactosides and has b e e n s u g g e s t e d to be a detoxifying e n z y m e [25]. D a p D transfers an a m i n o moiety from g l u t a m a t e to N-succinyl-~-keto-a-aminopimelate [26]. It utilizes pyridoxal p h o s p h a t e as a coenzyme, as d o Fig. 2. The imperfect tandem hexapeptide repeat structure of eight bacterial proteins. The hexapeptide arrangement was revealed by locating those IG, LG, and VG dipeptide residues which are followed or preceded by lie, Leu, or Val at an [I,V,LHI,V,L] distance of six residues. Such dipeptides as well as each lie, Leu, and Val continuing this six-residue periodicity pattern is underlined (by a double line). (A) The Ssc protein of S. typhimurhan and UDP-N-acetylglucosamine acyltransferase (LpxA) of E. coil The overlapping regions of these proteins are 22.4% identical [19]. (B) Thiogalactoside acetyltransferas¢ (LacA) and serine acetyltransferase (CysE) of E. coll. and the nodulation protein NodL of R. leguminasorum. These proteins have been suggested to form a single 'bacterial acetyltransferase' family [20]. (C) The hypothetical proteins Yglm of E. coil and Tins of B. subtilis. These proteins are 43% identical [23]. (D) Succinyldiaminopimelate aminotransferase (DapD) of E. coll. The alignments were made by using HIBIO PROS1S. The 31-residue long region of highest homology between all the eight proteins [19] is marked with asterisks. Deletions are marked with ( - ). 254 the other aminotransferases. Accordingly, it is difficult to find any common functional denominator specific enough to give any clue why these proteins all resemble each other and have the long hexapeptide repeat regions with the common theme. No three-dimensional structures have yet been determined for any of these proteins. Besides LpxA, LacA, and DapD, five other proteins were found to have the hexapeptide repeat theme. While no conclusions can be made regarding to the hypothetical proteins (Yglm of E. coli and its Bacillus analogue Tms), CysE and probably NodL are acetyltransferases as is LacA, and S ~ / F i r A could well have a function related to LpxA. In addition to LpxA, at least three other acyltransferases participate in lipid A biosynthesis [18,27], but none of them have been characterized at the protein or DNA level yet. Studies in this laboratory arc in progress to define the role o f Ssc. Thc hcxapcptide repeat theme now found m i g h t f a v o u r t h e f o r m a t i o n o f /3-turns, as d o s o m e o f t h e previously d e s c r i b e d r e p e a t motifs. These include the [L,I,F]XGGXG[N,D]DX motif f o u n d in t h e p o r e - f o r m i n g R T X cytolysins o f G r a m - n e g a t i v e b a c t e r i a [3,28] a n d t h e Gly-, A s n - , a n d A s p - r i c h m o t i f s in several c a r b o h y d r a t e - b i n d ing p r o t e i n s o f G r a m - n e g a t i v e b a c t e r i a [5]. A l s o t h e Pro-rich t a n d e m p e n t a - to n o n a p e p t i d e m o tifs in p r o l a m i n s , R N A - p o l y m e r a s e 11, r h o d o p s i n , s y n a p t o p h y s i n , a n d synexin f o r m r e p e a t e d / 3 - t u r n s [29,30]. It h a s b e e n s u g g e s t e d t h a t c o n s e c u t i v e p - t u r n s m i g h t be o n e a d v a n t a g e n o u s way to b i n d c e r t a i n ions, ligands, o r s u b s t r a t e c l a s s e s [3,5]. During evolution, intragenic recombination b e t w e e n t h e r e p e a t s e q u e n c e s in t h e g e n e a n d t h e r e s u l t a n t v a r i a t i o n in t h e n u m b e r a n d arr a n g e m e n t o f r e p e a t s m i g h t p r o f o u n d l y alter t h e s u b s t r a t e specificity. R e g a r d i n g to possible evolut i o n a r y relations, it s h o u l d be n o t e d t h a t t h e genes encoding Ssc/FirA, LpxA, and DapD are all l o c a t e d at 4 m i n in E. coli c h r o m o s o m e , a n d very close to e a c h o t h e r [6,12,17,31]. ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d by G r a n t 1011749 f r o m t h e A c a d e m y o f F i n l a n d a n d by t h e Sigrid Juselius Foundation. REFERENCES [I] Cohen. C. and Parry, D.A.D. (199(I) Proteins 7, 1-15. {2] Lupas. A., Van Dyke, M. and Stock, J. (1991) Science 252, 1162-1164. [3] Welch, R.A. (1991) Mol. Microbiol. 5. 521-528. [4] Tan. L.K.. Shopes. R.J.. Oi, V.T. and Morrison. S.L. (19911) Proc. Nail. Acad. 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