AbstractTwo bacterial acyltransferases (LpxA of Escherichia coli, LpxD of E. coli and Salmonella typhimurium) have previously been shown to consist of a very unusual tandem-repeat structure with tens of repeating hexapeptides (24 hexapeptides in LpxA, 26 in LpxD). By sequencing LpxD of Yersiniaenterocolitica (a distant relative of E. coli and S. typhimurium within Enterobacteriaceae) as well as LpxA of S. typhimurium and Y. enterocolitica, and by analyzing the existing data on these enzymes of Ricketsiarickettsii, it was now shown that the hexapeptide repeat pattern is a very conservative property of these enzymes. Even though the overall homology (allowing equivalent amino acids) between the four proteins was only 59% in LpxA and 58% in LpxD, the homology in the first residue of each hexapeptide was 87% in LpxA and 100% in LpxD. Secondary structure prediction by PredictProtein server suggested a very strong beta strand dominance in all the hexad regions. Accordingly, LpxA and LpxD of various bacterial origins can now be regarded as structurally very unusual enzymes, largely consisting of hexad repeats

Härkönen, Taina

Tolvanen, Martti

Vaara, Martti

Vuorio, Riitta

Elsevier - Publisher Connector 

FEBS Letters 337 (1994) 289-292 
Gim 
LETTERS 
FEBS 13546 
The novel hexapeptide motif found in the acyltransferases LpxA and LpxD 
of lipid A biosynthesis is conserved in various bacteria 
Riitta Vuorioa,*, Taina H&-khen”, Martti Tolvanenb, Martti Vaaraa 
aDepartment of Bacieriology and Immunology, and “Department of Biochemistry, PO. Box 21 (Haartmatzinkatu 3), 
SF-00014 University of Helsinki, Helsinki, Finland 
Received 1 December 1993 
Abstract 
Two bacterial acyltransferases (LpxA of Escherichia coli, LpxD of E. coli and Salmonella typhimurium) have previously been shown to consist of 
a very unusual tandem-repeat s ructure with tens of repeating hexapeptides (24 hexapeptides in LpxA, 26 in LpxD). By sequencing LpxD of Yersinia 
enterocolitica (a distant relative of E. coli and S. typhimurium within Enterobacteriaceae) aswell as LpxA of S. typhimurium and E enterocolitica, 
and by analyzing the existing data on these enzymes of Ricketsia rickettsii, it was now shown that the hexapeptide repeat pattern is a very conservative 
property of these enzymes. Even though the overall homology (allowing equivalent amino acids) between the four proteins was only 59% in LpxA 
and 58% in LpxD, the homology in the first residue of each hexapeptide was 87% in LpxA and 100% in LpxD. Secondary structure prediction by 
PredictProtein server suggested a very strong beta strand dominance in all the hexad regions. Accordingly, LpxA and LpxD of various bacterial origins 
can now be regarded as structurally very unusual enzymes, largely consisting of hexad repeats. 
Key words: LpxA; LpxD; Acyltransferase; Hexapeptide r peat heme; Proteobacteria 
1. Introduction 
We have previously found a novel hexapeptide repeat 
theme in eight proteins [1,2]. These include LpxA [3], 
LacA [4], CysE [5] and DapD [6] of Escherichiu coli, 
LpxD of E. coli [7] and Salmonella typhimurium (gene 
formerly named asfir, or ssc) [8], NodL from Rhizobium 
leguminosarum [9], Yglm of E. coli [lo] and its analogue 
Tms of Bacillus subtilis [ 111, as well as Yerm of B. sphae- 
ricus [12]. Of these proteins, Yghn/Tms and Yerm are 
hypothetical and the others are enzymes. LacA functions 
as a thiogalactoside acetyltransferase, CysE as a serine 
acetyltransferase and the nodulation protein, NodL, is 
probably also an acetyltransferase. DapD is a succinyl- 
amino-pimelate aminotransferase. The two remaining 
enzymes, LpxA and LpxD, have more of their structures 
made with this periodically repeating hexapeptide motif 
than do the other proteins mentioned above. The repeats 
cover 55% of the total lenght of LpxA and 44% of that 
of LpxD [2]. LpxA and LpxD are essential, remarkably 
homologous [ 1,3,8,13] and the self-comparison shows 
that they both contain regions homologous with other 
parts of the same protein. Mutations in either of these 
enzymes cause the bacterial cell to become thermosensi- 
tive and to quickly die after a raise of temperature and 
also sensitize the bacteria to hydrophobic antibiotics 
*Corresponding author. Fax: (358) (0) 434 6382. 
[13-161. The functions of LpxA and LpxD are related 
as they are both involved in the synthesis of lipopol- 
ysaccharide (= LPS). LpxA is the first (UDP-N-ace- 
tylglucosamine acyltransferase) [3] and LpxD the second 
acyltransferase (UDP-3-O-[3-hydroxymyristoyl] glucos- 
amine N-acyltransferase) [ 17.181 in the biosynthesis of 
lipid A, the hydrophobic part anchoring LPS to the outer 
leaflet of the outer membrane. The codon usage for 
L?. coli 1pxA and 1pxD is typical for a lowly expressed gene. 
Because about half of the LpxA and LpxD proteins 
are made of hexad repeats, we were wondering whether 
this motif is essential for the function of these proteins. 
If it is, the theme is expected to be conserved in various 
bacteria. To test this, we sequenced IpxA and 1pxD genes 
from Yersiniu enterocolitica and lpxA from S. typhi- 
murium and compared their deduced amino acid se- 
quences with those already known (i.e. lpxA and lpxD of 
E. coli (=firA) and IpxD (= ssc) of S. typhimurium). We 
also modified the original consensus equence which we 
have described before [1,2], to find the minimal back- 
bone covering this theme, and tested whether it could be 
able to find similar hexapeptide repeats in additional 
bacterial proteins. 
2. Materials and methods 
2.1. Bacterial strains, plasmids and phage Ml3 
The bacterial strains were rough derivatives of S. typhimurium LT2 
(SH.5014) [S] and E enrerocolitzca (EH902) [19]. E. coli strains JMl05 
0014-5793/94/$7.00 0 1994 Federation of European Biochemical Societies. All rights reserved. 
SSDZ 0014-5793(93)El463-V 
290 
[ZO] and TGl [21] were used as cloning hosts and pUCl9 was used as 
the plasmid vector. IpxD of 1: enierocolitux was sequenced from 
pUCHSll5 ([19] and from its derivative pUCHSll4, which carries a 
smaller genomic insert (the PstI-BarnHI fragment of pUCHSl15) in 
pUCl8. Besides plasmid sequencing, also M 13 mp8 and mp9 clones [22] 
were used in sequencing IpxA of S. rJ@~~rium. Bacteria were grown 
under aeration at 30 or 37°C in LB broth with tetracycline f 12.5 Bgfml) 
or ampicillin (100 .&ml) when necessary. 
2.2 General DNA techniques, cloning of ~xA 
Isolation of chromosomal DNA, preparation of hybridization 
probes, their radiolabeling, Southern blotting, cloning, sequencing and 
other DNA manipulations were carried out as described in [19]. After 
nick translation, free label was separated from the labeling mixture with 
Nuctrap push columns (Stratagene, La Jolla, CA). To clone IpxA of S. 
typhimurium and Z enterocolitica, probes were prepared from 
pUCHS16 [8] and pUCHSll5, respectively. The labeled probes de- 
tected a 5.0 kb ~~~I-fra~ent from the chromosome of SH5014 and a 
2.5 kb ~.s~i-~g~l-fra~ent from the chromosome of EH902. These 
fragments were ligated to pUCl9 and the resultant plasmids were 
named pRV40 and pTH2, respectively. 
2 3 Computer analyses 
The conserved motif [(I,V,L)GXXXX],(I,V,L) was used to screen all 
bacterial amino acid sequences in SwissProt (release 25.0, April 1993) 
with the Findpatterns program allowing two mismatches. The multiple 
alignments were made with the Pileup program using default parame- 
ters (both programs provided by the Genetics Computer Group, Pro- 
gram Manual for the GCG Package. version 7, 1991). The equivalent 
amino acids in these alignments were: A,G,S,T.P; E,D,N,Q; H&R; 
I,L,M,V; F,Y,W C. The ProSite 1231 database (ver. 10.1) was searched 
for other consensus patterns of the proteins containing this hexapeptide 
repeat theme. The absence of three-dimensional structures for any of 
these proteins was confirmed in the Brookhaven Protein Data Bank 
(PDB. release of Jan 1993). Secondary structure predictions were per- 
formed with the Heidelberg neural network prediction server [24] (data 
and software as on Ott 7, 1993). The PredictProtein server was given 
three different sequence alignments: LpxA and LpxD sequences with 
four aligned sequences in each group, and all eight sequences together. 
2 4. Nucleotide sequence aecession numbers 
The EMBL accession umbers are: 225462 for the IpxA gene from 
S typhimurium SH5014 and 225463 for the sequence containing /p.uD 
and Ip.uA from Z enterocoliticu EH902. 
Ftg. 1. The aligned LpxA sequences of E eoh (ECOLPXA). S ry- 
phlrnur~~ (STMLPXA), Z enterocolit~eu (YECLPXA) and R. rick- 
ettsii (RRILPXA). The numbering is accordmg to the E. coli sequence. 
Ile, Leu, Val and Met residues conforming to the six-residue periodicity 
are boxed. The gaps are marked with a dash (-) and identities with a 
period (.). 
ECOLPXD 
STMLPXD 
YECLPXD 
RRILPXD 
ECOLPXD 
STMLPXD 
YECLPXD 
RRILPXD 
ECOLPXD 
STMLPXD 
YECLPXD 
RRILPXD 
ECOLPXD 
STMLPXD 
YECLPXD 
RRiLPXD 
ECOLPXD 
STMLPXD 
YECLPXD 
RRILPXD 
ECOLPXD 
STMLPXD 
YECLPXD 
RRILPXD 
ECOLPXD 
STMLPXD 
YECLPXD 
RRILPXD 
ECOLPXD 
STMLPXD 
YECLPXD 
RRILPXD 
R. Vuorio et al. I FE3S Letters 337 {I9941 289-292 
2 39 
PSIRLADLAQQLDAELHGD--GDIVITGVASMQSAQTGHI 
. . . . . . . ..E..... . ..--..............T.... 
. . . . . . . . . . . . . . QV...--..L....I...H..EPSQ. 
MVSSNPYKNLGPRK.TAIIDF.HDITAPPKIE..A,HDIKIL.E.SPND. 
40 87 
TFMVNPKYREHLGLCQASAWEPTQDDLPPAKSAALVV--XNPYLTYARMA 
. . . . . . . . . . . .._____... . ...*.*.. . . . ..--..... ..‘., 
..LS.SR.Q.Q.AT.N.G...L.EA....C.......--....... . . 
S LS.. .S.F.KTTK.A.CXVPKNFTGE.NPNTVLLHAQ.S.FA.GKLI 
174 219 
ADGFGYANDRGNWVKIPQIGRVI 
308 34i 
QPNKVWRKTARL~~NIDDMSKRLKSLERKVN~QD 
. . . . . . . . . . . . ..AI........ 
,..... ..GIN....AI...IDKE 
I.IMD.HRQSIIMKQL------..TSNS LKK 
Fig. 2. The aligned LpxD sequences of E coli (ECOLPXD), S. TV’- 
phimurium (STMLPXD). I! enterocolrticu (YECLPXD) and R. rick- 
cttsri (RRILPXD). The six-residue periodicity and gaps are marked as 
m Fig. 1. The mature E co/i protein does not contain the initiation 
methionine [7]; this restdue is omitted from the other enterobacterial 
sequences, too. 
3. Results and discussion 
We have been impressed by our recent finding that the 
acyltransferases LpxA and LpxD of E. coli and LpxD of 
S. t~~p~imur~um are, to a remarkable extent, built from 
repeating hexapeptide blocks. To see how universal a 
property this is for enterobacterial LpxA and LpxD pro- 
teins, we determined the sequence of LpxA of S. ty- 
phimurium and those of LpxA and LpxD of I: entero- 
colitica, a phylogenetically distant relative of Escheri- 
chiae and Salmonellae. Also the sequences of 1pxA and 
ZpxD of ~icket~ia rickettsii have been determined re- 
cently [25], which enhances the available info~ation. 
These four species all belong to the eubacterial group 
proteobacteria (rickettsiae to the a subdivision, enteric 
bacteria to the y subdivision) [26]. 
Each of the LpxA (Fig. 1) and LpxD (Fig. 2) proteins 
R. Vuorio et al. IFEBS Letters 337 (1994) 289-292 291 
now known display a hexapeptide repeat pattern. Each 
hexad starts with Ile, Leu, Val (or their equivalent [27] 
Met), and, as also noted previously [2], the second posi- 
tion is often occupied by Gly, the fourth by Gly, Asn or 
Asp and the fifth by Val or Ala. There are 24 hexads in 
LpxA and 26 hexads in LpxD of the three enterobacterial 
species; the corresponding numbers for R. rickettsii are 
19 and 25, respectively. The identity between the entero- 
bacterial LpxA proteins is 80% and between all four 
LpxA proteins, 35%. The corresponding similarities (al- 
lowing equivalent amino acids) are 89% and 60%, respec- 
tively. The identity percentages between the LpxD pro- 
teins are 82% (enteric bacteria) or 30% (all four bacteria); 
the corresponding similarities are 94% or 60%, respec- 
tively. 
served. These residues can be regarded as backbone res- 
idues. 
The degree of conservatism within different regions of 
LpxA and LpxD is shown in Table 1. Regarding LpxA, 
it is obvious that the only notably conserved amino acids 
in the four bacteria are placed as the first and second 
residue of each hexad. As for LpxD, the preserved loca- 
tion is the first residue of the hexads (there is also a short, 
10 residues long, highly conserved region from position 
215 to 224 between two hexapeptide regions). The other 
parts of LpxA or LpxD do not show any preferred con- 
servation. 
Accordingly, it is now clear that the hexapeptide re- 
peat pattern is very characteristic for the LpxA and 
LpxD proteins of all the studied bacteria. Even though 
the overall sequences of these proteins from different 
bacteria do not appear to be unexceptionally conserved, 
the hexad arrangement and the first residue (in LpxA, 
two first residues) of each hexad are extremely con- 
We then simplified our original consensus equence [l] 
to make it cover the hexad repeat motif as a universal 
theme. The final motif is [(I,V,L)GXXXX],(I,V,L). By 
this sequence, using the Findpatterns program and al- 
lowing two mismatches, we found most of the proteins 
which we have previously reported to contain a homolo- 
gous theme. Only Yglm and DapD were left outside. The 
search found all the members of the suggested ace- 
tyltransferase family [23], i.e. CysE (from E. coli and S. 
typhimurium), LacA, NifP (a putative serine acetyltrans- 
ferase from Azotobacter chroococcum) [28], and NodL 
(from Rh. leguminosarum and Rh. meliloti) [29]. The 
search also detected the chloramphenicol acetyltrans- 
ferases (CATS) of the E. coli transposon Tn2424 [30] and 
Agrobacterium tumefuciens [31]. These two CATS have 
no similarity with other chloramphenicol acetyltrans- 
ferases. Finally a j?-glucosidase from B. polymyxa ([32] 
gene name: bglA) as well as a 24.3 kDa hypothetical 
protein from enterobacterial antibiotic resistance plas- 
mid Rl [33] was found to contain this motif. The p- 
glucosidase active site and the hydrolase consensus pat- 
tern are situated outside the hexad theme. To summarize, 
the hexapeptide motif can now be stated to be character- 
istic of certain families of acyl- and acetyltransferases, as
well as of some other proteins. 
Periodicity repeat themes are very rare in enzymes (for 
known examples, see [2]). It is plausible to expect that the 
hexad regions of LpxA and LpxD form a structural 
(non-catalytic) part of the protein. We employed 
PredictProtein program to predict the secondary struc- 
Table 1 
Conservatism in different regions (hexapeptide vs. non-hexapeptide) of the known LpxA and LpxD proteins, shown as the missmatch percentage 
in the aligned amino acid sequences 
Missmatch %b in 
LpxA” LpxD” LpxA LpxD 
Entire protein 41 42 
Non-hexapeptide regions 
Region A ( o- 1) ( 2- 99) 100 41 
B ( 68 85) (178202) 44 36 
C ( 98-110) (215-224) 46 0 
D (177-262) (291-341) 52 55 
Total (A-D) 118 residues 184 residues 51 45 
Hexapeptides (abcdef) 
Residues m position a 13 0 
b 17 38 
C 38 35 
d 54 58 
e 33 46 
f 46 58 
Total (a-f) 26x6=144 26x6=156 33 39 
a The amino acids in the aligned LpxA and LpxD (see Figs. 1 and 2) are numbered according to the E. co/i sequences. The locations of non-hexapeptide 
regions (residue positions) are shown in parentheses; the rest of the proteins is built from repeated hexads. The non-aligned regions in the amino 
(LpxD) and carboxy (LpxA) termini of R. rickettsir sequences, as compared to the E. coli proteins, are omitted. 
b Indicates the percentage of non-conservative amino acid substitutions from the total number of amino acid residues. Conservative substitutions 
are defined in Section 2. 
292 
ture of these proteins and found a clearcut predominance 
of beta structure in the hexapeptide region. In case of 
LpxD the PredictProtein server judged this region to 
consist solely of beta strands and of loops connecting 
them. The prediction for LpxA shows four short, less 
certainly predicted alpha-helical segments in addition to 
the dominating beta structure, whereas the combined 
prediction of the eight sequences i all-beta in this region. 
In the C-terminal region both LpxA and LpxD show 
alpha helices predicted with a high reliability. In the 
N-terminal 110 amino acids of LpxD there are two 
strongly predicted alpha-helices as well. 
Interestingly, all the known defective alleles of lpxD 
(i.e. ssc-1, omsA,jirA2OO,JirA201) have point mutations 
in the hexad region or (in SSC-1) in the residue immedi- 
ately flanking the hexad region [8,13,14]. Unfortunately, 
there is no crystallographic data on the structure of any 
of the proteins with the hexapeptide repeat theme. It 
would now be very interesting to determine the three- 
dimensional structures of LpxA and LpxD, including the 
mutant proteins of the latter. 
Acknowledgements, We thank B. Kuusela for excellent echnical assis- 
tance. This study was supported by Grant 1011749 from the Academy 
of Finland and by the Sigrid Juselius Foundation. 
References 
[l] Vuorio, R.. Hirvas, L. and Vaara. M. (1991) FEBS Lett. 292, 
90-94. 
[2] Vaara, M. (1992) FEMS Microbrol. Lett. 97, 249-254. 
[3] Coleman, J. and Raetz, C.R.H. (1988) J. Bacterial. 170, 12688 
1274. 
[4] Fowler, A.V.. Hediger, M.A., Musso, R.E. and Zabin. I. (1985) 
Biochimie 67, 101-108. 
[5] Tei, H., Murata, K. and Kimura, A. (1990) Biochem. Biophys. 
Res. Commun. 167. 9488955. 
[6] Richaud, C., Rrchaud, F., Martin, C., Haziza. C. and Patte, J.-C. 
(1984) J. Biol. Chem. 259, 1482414828. 
[17] Raetz, C.R.H. (1993) J. Bacterial. 175, 574555753. 
[l8] Kelly, T.M., Stachula, S.A., Raetz, C.R.H. and Anderson, M.S. 
(1993) J. Biol. Chem. 268, 1986619874. 
[I91 Hirvas, L., Koskr, P. and Vaara, M. (1991) J. Bacterial. 173, 
1223-1229. 
PO1 Yanisch-Perron, C., Vieira, J. and Messing. J. (1985) Gene 33, 
103-l 19. 
WI 
P-21 
1231 
1241 
~251 
Gibson, T.J. (1984) Ph.D. thesis, Cambridge University, Cambr- 
idge, UK. 
Messing, J. and Vieira, J. (1982) Gene 19, 2699276. 
Bairoch. A. (1993) Nucleic Acids Res. 21. 3097-3103. 
Rost, B. and Sander, C. Nature 360. 540. 
Shaw, E.I. and Wood, D.O. (1993) Genbank Accessron No. 
L22690. 
WI 
1271 
WI 
P91 
[301 
[311 
[321 
[331 
Woese, C.R (1987) Microbial. Rev. 51, 221-271. 
Hrggins, D.G. and Sharp. P.M. (1988) Gene 73, 237-244. 
Evans, D.J.. Jones, R., Woodley, P.R., Wilbom, J.R. and Robson, 
R.L. (1991) J. Bacterial. 173, 545775469. 
Nedelcho. B. and Kondorosi, A. (1992) Plant Mol. Biol. 18.8433 
846. 
Parent. R. and Roy, P.H. (1992) J. Bacterial. 174, 2891-2897. 
Tennigkeit, J. and Matzura, H. (1991) Gene 98, 113-l 16 
Gonzalez-Candelas, L.. Ramon, D. and Polaina, J. (1990) Gene 
95, 31-38. 
Bravo, A.. de Torrontegui, G. and Diaz. R. (1987) Mol. Gen. 
Genet. 210, 101-l 10. 
R. Vuorro et al. IFEBS Letters 337 (1994) 289-292 
[7] Dicker, I.B. and Seetharam, S. (1991) J. Bacterial. 173, 334-344. 
[8] Hirvas, L., Koski, P. and Vaara, M. (1991) EMBO J. 10, 1017- 
1023. 
[9] Hayo, C.J., Cremers, C., Spaink, H.P., Wijfjes, A.H.M.. Pees, E., 
Wijffelman, CA., Okker, R.J.H. and Lugtenberg, B.J.J. (1989) 
Plant Mol. Biol. 13, 1633174. 
[lo] Walker, J.E., Gay, N.J., Saraste, M. and Eberle, A.N (1984) 
Biochem. J. 224, 799-815. 
[ll] Nilsson, D., Hove-Jensen, B. and Arnvig, K. (1989) Mol. Gen. 
Genet. 218, 565-571. 
[12] Monod, M., Mohan, S. and Dubnau, D. (1987) J. Bacterial. 169, 
340-350. 
[13] Dicker, I.B. and Seetharam, S. (1992) Mol. Microbial. 6,817-823. 
[14] Vuorio, R. and Vaara, M. (1992) J. Bacterial. 174. 7090-7097. 
[15] Galloway, S. and Raetz. C.R.H. (1990) J. Biol. Chem. 265,6394- 
6402. 
[16] Vuorio, R. and Vaara, M. (1992) Antrmicrob. Agents Chemother. 
36, 826829. 


The novel hexapeptide motif found in the acyltransferases LpxA and LpxD of lipid A biosynthesis is conserved in various bacteria 

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The novel hexapeptide motif found in the acyltransferases LpxA and LpxD of lipid A biosynthesis is conserved in various bacteria

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