Appl Microbiol Biotechnol (1990) 34:229-230

Applied Microbiology Biotechnology © Springer-Verlag 1990

Short contribution Highly homologous cyclodextrin glycosyltransferases from Bacillus circulans strain 8 and a strain of Bacillus licheniformis Hans Bender Institut for Organische Chemie und Biochemie der Universit~it Freiburg im Breisgau, Albertstrasse 21, D-7800 Freiburg im Breisgau, Federal Republic of Germany Received 20 July 1990/Accepted 27 July 1990

Summary. Alignment of the amino acid sequences of the cyclodextrin glycosyltransferases and their signal peptides from Bacillus circulans strain 8 and a strain of B. licheniformis revealed high homology with 90.8% and 85.3% of identical amino acids. Of the codons of the CGTase genes and of the leader sequences, 45.2% and 47.0%, respectively, proved to be identical. A I ~AT CCG GAC ACT GCT GIG ACC.AAC AAA CAG AGE TIC iGT ACA GA[ 6[G A|C TAC CAA 2 '*' G" "[ "'A "'* "C "* "'* "G "" ' A * * * ' " ' * *'G " ' * '*C " * ' " ' [ * ' *

GIA I l l "'[ • l~l 'I"

The fl-cyclodextrin glycosyltransferase gene [1,4-Ot-Dglucan: 1,4-C~-D-glucopyranosyltransferase (cyclizing), EC 2.4.1.19; CGTase] of Bacillus circulans strain 8 has been cloned in Escherichia coli, and the amino acid sequence of the encoded polypeptide (684 residues with mol. wt. = 74416) has been elucidated at the D N A level (Nitschke et al. 1990). The enzyme showed 72.2% and ACT 6AA CAG IAT TTG ACG GGG AAT G6C GAT CCA GAT AAC CGG GCC AAA ATG CC[ TCG *'C ' % ' * ' * * ' C " * ' I * " ' " C ' * ' ®'C * ' G ' " "' *** 'G' '** "" "* *'C

ACG GA| CGC T|I TTG fiAC GGC AA[ CCC TCC ~AC AAC CCC ACC 6GA GCC GCT " ' " " ' C ' ' A * ' ' C ®* " l "[ *°C " A " " '" '*' "[ '" **G " I '*C

I I C ICC AAA |CC AC| ACA GC[ | | ! AAC GTC A|A AG| AAA CIC GCG CCT CTG CGC AAA * ' * ' ' " * * ' **G ° * " ° ' ~ " ' G **C * ' " ~*G " ' C * ' C "*G " ~ ' * A " * ' ' * * * ' ' " ' *

GA[ CCC ACA IGC ACC AAI I I G AAG CTG TAC ICE GGC GGA GAC |CG CAA GGG ITA ' * ' "G' " ' G " ' ' " " "C C'* ''" "*' '" *" *'| "'G '" "" " G **A " G

ICC AAT CCG GCC A [ | GCC |AC GG[ TCC ACA CAG CAG CGC TGG ATT AAI AAT GA[ GTA * ' * ' * C * * * * ' G * ° * ' * * = * ' " ' ' * * " ®'¢ ' ' " ' ' ' " ' * * ' " "~C " C ~ " ' * " ' ' '

'*'

A1C AAC AAA AIC AAC GAI AAC TAT ITC ACT CA| CIG GGT GTC ACA CCG ITG |GG ATC G'" '*" '*" '*' "[ '*C *~' " ' " " " °'" "'C I'" "" *" " G " ' C C'C ' " '" • ICC CAG CCI GIC ~AA AAT A ] | TIT CCG ACG AIC AAC TAT A~C GG[ ~TA ACC AAT ACT ' ' * * * * ' * ' **G * * " **~ ' ' ' "~C * ' I * ' C ' ° * ' ' ' '*C *'' ..o ... *** ... ''A

CIT ICC ACG TCI ~CA A~C A I I ACA GGG ~TA AGC ACT ICC CTG CC[ ACA G~C TCA TAC " C A'G " " £ ' A A'C " ' [ " ' C ° ' | AAC C ' ~ ' A [ ° ' G " ' G " ' | " ' A T ' " " ' A A~C " ' T

-GCC IAC CAC GGC IAC TCG GC[ C~A GAC l i e AAA AAG ACC AAI CCG 1AC TIC 6CC ACG *'A ''G ''I m*. , . ~ * , , , . , . . * * , . " ' l " ' * "°A " C '" '*! '" "" "! ""

ACG GAI 6 [ 6 C I | GGC CGG GIG C[G AAC 6GA AA] A~C AIC ~CC ]CC ACG AAI GG[ AGC "'C ' " ''° "C "° °°C " " °°" "" "" "C "" "" " G " ' " "G! GGA " C " A I

RIG GCG CAC I l l " * * A*C ' * I ' "

All "'"

CAG ~AE CG| A l l ACG ACG GC[ CAT GCC AAA CGC ATC RAG AT[ G|C '" * * ! " * ' G'A " C ' * E " ' C ' " *°G ' " "" ° * ' " ' k " C A*T

lk[ **'

6111A[ ~'C " *

GAG C~[ AAA ] [ C G6C AAG A6C GIA GCC GIT GTC GCG GIA AAC CGC AA[ " * * " C **G " l "* '*A "" "l *'" "'C "[ '*C **T . . k . , . , . ,

AAC AAC | I C ACC C [ | GC[ GC6 6G| GCA ACG GCA GTA |GG CAA ]AC ACA ACT GCC ] C [ IC" * ° ° ' ' G * ' C ' * ° " A * ' A ~ ' l * ' C " [ "[ " * ' H . * . [ ' * G G'A AGI

A [ | CAC TII GCA CCG AAI CAC ACC IC1 CCA GCG A|G GAA ACC GAC ACC 1C[ |11 6CC ° ' " " ' 1 " ' C '*G ' ' A ' * C " ~ i "~G ° ' C " | *'C ' * " ' ' ' "' " ' I ' * ' *'C ' ' C " ' *

GAA ACA ACA CCA ACC A|C GG[ CAT G I | GG! CCG G I [ A|G G6G AAA CCC GGT AA~ GIG ' * ' * ' G * ' ~ ' ' ° * ' ' ' * " ' ' " ' ' C " ' " ~'C ' * | ' ° k * ' * ' ° [ ' * " " ' A * * * " ' C ' ° C

GAA AAT GGC AGA CIG IAC 6A[ AAC GGI ACA C|G GIA ~GC fifil I A I ACC AA[ CA[ ACC ' ' C * ' ~ * ' ' "A* * ' ° * ° ' ~ ' ° ° ° * ' C "AC " * * ' ' | *°* °*l; ' ' C ' * ' * ° ' ° ' * " * *

CIG ACA AIT ~A| GCC CGT GGA |TC 6 f ~ TCG ACG AAA GGC ACG 61C TAC TIC CGC ACT "I " ' C ° ' C " ' C ° ' A " C " ' G ' ' " ' ° 1 ~'C G'C ' ' ' "T "'C "'" "" "" "'I "A

ARC G~G IAC TIC CAI CAC A~i CCC G(;C ICC GAC |IC |C| |CC C|G ($~G ARC ~{;C AIC *°' '*A ''| '" "C "* ''* '" '*' *" °'I ''' " C A'T ' ' I '*" *'1 *'' '*[

ACA ~C6 GIT ACC GCA GCA GCG A l l ~'" "C '" "G "C I" "C ""

|AC AAA AAC C[G TA| GAC CGT GCC CAC [ I C AAC CAC AA| AAI (;CG ACC AIC GAC AAA "'* *'* "'' ''C ''C "'I "" " ' ' ' * T G'G " ' [ " ' ''' ' ' C AGC ' ' G ' ' * * ' I ' C "

AIC CCT TCC GIT GCC GC! GGC AAC TAT C,CA GIC AAA GT| GCG GCA AGC GGG GTA AAC ''l ' ' k C'A ' ' A " ' A "C~C " T G ' " ' " "" " G " ' " " ' A " T " C ' A [ " ' 1 "°G " "

TAC TIC AAA ~A| ~CG ATC AAA CIG |GG CTT GAT AIG GCC GF| GAC GCI h l | CG(; GIG *'| ''* "° " C " * ' 1 ' ' *'G " " ''' . . s . . 1 1 ° . , . . *'G ° ~ ' " C ' * ~ " l °*C

ACC ART CC4 TAC ARC ARC |TC kCC ATC CfG AC[ CGC CAI CAG GIC ACC GT~ CGC TTC '" "'C "C "T '" G'! "" °" "" ~ ' [ 'GC ' " "" "" " A T°G "°G " G " "

(;A| GCG (;IG AAA CAI AIG CCi CIG I~(;I I(;G CAn AAG Af;C IGG ~tG ICC ICC ~IC IAC " ' * " ' ' ' ' C " ' G ' ' C * ' " . . . . AG *'C " ' "G ''' 'A' ''' "'" °'A °'I ''* ''[

6IC I;IA AAC AA| CCG ICC ACA ACG C|T CCA CAG AAC CIC TAT TIG ACA GGC AAC G|A ' ' ' A'C " ' l ' ' " " ' C A ° A ° ° | G' ° " G ~'C G ' " ' ' ~ A m. ' ' C C m' ' ' " H . . . . "'G

~CA CAC AAA CCC GIG TIC ACT I l l GGA GAA |GG TIC [ I G GGA ICA CC! CCA ICC GAT °'* '" " ' G " G " A " ' 1 " C **C **C ' " "" "'* C'" "' "T "" *" C'" "'"

CCC ~AG C | | G[~C ARC IGG AGC ACC GCT TCG AC[ GCC ATf CGA CCC GCA |TC ART CA~ l'' ''A ''C ''I ' ° " " ' ' ' C " * ' A " * * G'A G ' ' l ' ' "" "'" '°G ''I ''[ ''' "'"

GCA GA| AAC ACG GAT l | [ ''G ''' *°' ''A ''* ''"

C|C A l l ,"" "C

GCT AAC AAG I C [ ~Gl AIG AGC C|6 CIC GAC TIC CGC I 1 | ' * C " ' | G'A ' ° ' ' ' * ' ' " " l I * ' 'CF ° ' I ' * ' " * | " C

ACG IC| IGG GAA ~AT ACA CAG AT( AAA GIA ACC "I "'A "'" "'" ''C "'T '" "'C ''" "'C "'"

CAT CAA |AC CC& ACC 'IGG TAC TA| GAT GIC AGC GTA CCG GCA ~GC AAA CAG " ' C GCC " ' " ' ° G " I "" "[ "'" "C "'A "'[ "'I "'" "'C "G "" '°"

AAC ICT GCG GIG CG| AAT GIG I1C CG| GAC ~AC ACG 1CC AAC AIG [AC GC| CGT GAI " 1 **G * ' I '*C " C " C ' " '" "G **I "* "C '" '** '°' ''" "'G "* *"

CIG GAG TIC AAA TIT ITC AAG AAA AAC 6GT TCA ACG ATT ACA TGG GAA AGC GG[ ICT "'G " C " ° '" "" '" ~'C G'T ' ° C " " "'G '" " " G'T " A " C '" "'A '"

ICC AlE h i [ ~AC ACC ACA CCI ACG ~AC ]AC ARC CAR GIG ARC CA! CAG GIG AC~ TIC "'° '" C*G "CG GC! " G " A G'A " ' ! ' " " ' f " ' G " ' * ° ' [ '*C " A "*C " ' ~ " "

AAC CAC ACA TIC ACT ACA CCA GCG AGC ~CA ACA GCC ACC GT[ ACG GTG AAC ]GG CAG *.! H. ''C "'T '*A ''' " ' G A"~ " ° ! " ' l ' ° T " ' " ' ' A " ' A " ' " A ' A " ' " " ' " " ' A

A]C GAC ARC CA~ GA| A]G ~A[1C~| ] I C AAA ACA AG! GCG G|C ARC AA[ CCC CGT CIG "1 '*' °'" "** "*' '" '*C . h . . * * . . *'C ' ' " " [ " G ' ' " '°C ' * " * ' ' ° ' ° CAA CAi$ r ~ l ° ' 1 1°° " ' "

IIG GCC I1C ACA I l l l AI:I |CA CI~I Gfi| G|A CC| 6CC AIC TAC I A I ~C[ C" .°" "° "'(~ C** " C ' * ' " C * ' C ' ' ' "*' '~* '*" "I "1' °'1

B 1 ATC l i t 2 *'~ "''

CAA A|G ~CC AAA CCC GCA TIC C]C AGC ACC ACA CT~ ACC CTC GGC TIG CIT " T ' ~ ' '°G " ' A " ' ~ l" A °" C "° " " "'° "'" ''" "" °'' °I! C'' "'

GCC GGC AGC (;CC C1G CCG I | C ITG CCA GC[ ~CC ~CT ~IA ~AC GCC "'' ''' "T ''A I'" '°T °'" C'" "'! ''° °'° °'A A'I "'l '''

Fig. 1. Alignment of the nucleotide sequences of the CGTase genes (A) and the leader regions (B) from Bacillus circulans strain 8 (1) and B. licheniformis (2): *, identical nucleotides

230 74.6% homology with the CGTases of the alkalophilic Bacillus sp. strain 1011 (Kimura et al. 1987) and strain F-2 o f B. circulans (Nishizawa et al. 1987), respectively,

which are the closest relatives so far. Comparison o f the primary structures o f the CGTases from B. circulans strain 8 and a strain o f B. licheniformis (Hill et al. 1990) revealed very high homology with 90.8% o f identical amino acids. Highest homology with 94% of identical residues is noticed for t h e region of the polypeptide corresponding to the N-terminal domains A and B, and lowest homology with 70.7% o f identical residues for a stretch of the polypeptide (position 439-480) corresponding to domain C, which is distant from the active site ( H o f m a n n et al. 1989). Both CGTases contain conserved deletions at positions 471 and 571, and an insertion at position 621, when compared with the other bacilli CGTases. The homology is not restricted to the CGTases, but extends to the signal peptides (34 residues), where 85.3% o f the amino acid residues are identical. From the alignment of the nucleotide sequences it becomes evident that 45.2% of the codons o f the CGTase genes, and 47.0% of the codons o f the leader sequences are identical (Fig. 1). Of the codons of the CGTase genes and the leader sequences, 48.4% and 47.0%, respectively, contain merely one nucleotide exchange, resulting mostly in synonymous codons, and in 19 and 3 codons with altered codon specificity. Only seven codons are exchanged completely. Besides the highly homologous CGTases o f the alkalophilic bacilli strains 1011 (Kimura et al. 1987) and 38-2 (Horikoshi 1988), which are likely to be closely related organisms, the enzymes o f B. circulans strain 8 and B. licheniformis are an example o f highly homologous proteins from different species, which is o f taxonomic and evolutionary interest.

Since the species B. circulans proved to be genetically very heterogeneous [several homology groups with 37-54 mol% G + C of D N A have been established (Nakamura and Swezey 1983; Fahmy et al. 1985)], the G + C content o f the CGTase genes can scarcely serve as a taxonomic criterion: 50.8 mol% G + C are determined for the CGTase gene of B. licheniformis, and 49.5% mol% G + C for that of B.. circulans.

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