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Protein

Science

(1997 ), 6301 -523. Cambridge University Press. Printed in the USA.

Copyright

0

1997

The

Protein Society

REVIEW

Subtilases:

The superfamily of subtilisin-like serine proteases

ROLAND J. SIEZEN

A N D

JACK A.M. LEUNISSEN2

Department of Biophysical Chemistry, NIZO, P.O. Box 20, 6710BA Ede, The Netherlands

CAOSKAMM Center, University of Nijmegen, Toernooiveld, 525ED, Nijmegen, The Netherlands

(RECEIV ED ugust 22, 1996; ACC EPTE Dovember

5 ,

1996)

Abstract

Subtila ses are memb ers of the clan (or superfamily) of subtilisin-like serine proteases. Over 200 subtilases are presently

known, more than 170 of which with their complete amino acid sequence. In this update of our previous overview

(Siezen RJ, de Vos WM, Leunissen JAM, Dijkstra BW, 1991,

Protein

Eng 4719-731), details of more than

100

new

subtilases discovered in the past five years are summarized, and amino acid sequences of their catalytic domains are

compared in a multiple sequence alignment. Based on sequence homology, a subdivision into six families is proposed.

Highly con served residues of the catalytic domain are identified, as are large or unusual deletions and insertions.

Predictions have been updated for Ca*+-bindingsites, disulfide bond s, and substrate specificity, based on both sequence

alignment and three-dimensional homology modeling.

Keywords:

homology modeling; sequence alignment; serine protease; subtilase; subtilisin family

Serine endo- and exo-peptidases are of extremely widespread oc-

currence and diverse function. Many distinct families of serine

proteases exist; they have been grouped into six clans (Rawlings

and Barrett, 1994; Barrett and Rawlings, 1995), of which the two

largest are the (chymo)trypsin-like and subtilisin-like clans. These

two clans are distinguished by a highly similar arrangement of

catalytic His, Asp, and S er residues in radically different PIP (chy-

motrypsin) and a @ (subtilisin) protein scaffolds.

In 1991, we presented a review of over 40 members of the

subtilisin-like serine proteases, termed sub tilases, which occur in

Archaea, Bacteria, fungi, yeasts, and higher eukaryotes (Siezen

et al., 1991). The mature enzymes were found to contain up to

1775 residues, with N-terminal catalytic domains ranging from

268 to

5 1

1 residues, and signal and/or activation-peptides ranging

from 27 to 280 residues. Several members contain C-terminal

extensions, relative to the subtilisins, which display additional prop-

erties such as sequence repeats, Cys-rich domains,

or

transmem-

brane segm ents. From four known crystal structures and a multiple

alignment of 40 known amino acid sequences, a core structure was

predicted for the catalytic domain of all subtilases, together with

the variations that are allowed in the main-chain length as a result

of insertions and deletions (Fig.

1).

Nineteen of these core residues

were found to be highly conserved, 10 of which are glycines.

Predictionswerealsomade for subtilases of unknown three-

dimensional structure concerning essential conserved residues, al-

Reprint requests to: Dr. Roland J. Siezen, Department of Biophysical

Chemistry, NIZO, P.O. Box 20, 6710BA Ede, The Netherlands; e-mail:

[email protected].

lowable substitutions, disulfide bonds, Ca2+-bind ingsites, substrate-

binding site residues, ionic and aromatic interactions, and surface

loops. Based on these predictions, strategies for homology mod-

eling and protein engineering were developed and implemented,

aimed at modulating either stability, catalytic activity, or substrate

specificity (Siezen et al., 1991, 1993, 1994, 1995a).

Since 1991, more than 100 new subtilases have been discovered,

and these are now included in this updated review. In addition to

many new enzymes from micro-organisms, numerous members of

the subtilase superfamily have now also been identified in various

eukaryotes such as slime molds, plants, insects, nematodes, mol-

luscs, amphibia, fish, mammals, and even in a catfish virus.

Structure-based alignment

The coordinates of subtilisin BPN, subtilisin Carlsberg, thermi-

tase, and proteinase K were used previously (Siezen et al., 1991)

to determine the core of structurally conserved regions (scrs;

Greer, 1990) and the common secondary structure elements, as

analyzed with the DSSP program (Kabsch and Sander, 1983). This

core of about 190 residues contains virtually all of the common

a-h elix and &strand elements, including the active site residues

D32, H64, and S221 (Siezen et al., 1991). Slight adjustments to

thesecore regions have now been incorporated (core ABC in

Fig. 2) based on a recent spatial superpositioning of seven struc-

tures that also included mesentericopeptidase, Savinase, and

Es-

perase (Heringaetal., 1995); topologically equivalent residues

were defined as those that have Ca-atom distances of less than

2.0 A . The variable regions (or vrs) nearly always correspond to

501


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502

R.J. Siezen and J.A.M. Leunissen

A

connecting loops between helices and strands and generally lien

the external surface of the protein (Fig. 1).

When only the subtilisin BPN', subtilisin Carlsberg, and ther-

mitase structures were superimposed the number of structurally

equivalent Ca atoms increased to over 230or about 85% of allCa

atoms), which we refer o as the extended core (core AB n

Fig. 2). This distinction between core and extended core scrs is f

relevance

for

homology modeling, because the superfamilyf sub-

tilases can be subdivided into several families (see below).

Identification of subtilase supetfamily members

An extensive search of scientific literature and databases (EMBL,

Genbank, Swiss-Rot) was performed o dentify new subtilisin-

like serine proteases, using the programs BLAST (Altschul et al.,

1990), TFASTA, and FASTA (Pearson and Lipman, 1988). Con-

sensus sequence segmentsf 20-40 residues around he active site

residues D32, H64, and S221 were used or this purpose; different

consensus segments were obtained or different subtilase families

(see Fig. 2). Sequences from patent literature and databases are not

included because they represent synthetic or mutated genes encod-

ing engineered subtilases. The main results of these searches are

summarized n Tables 1 and 2. Further details, including reference

to 10 crystal structures, can be found in the EMBLlGenbank and

PDB databases using codes listed in the tables.

At present, over 170 complete and several partial amino acid

sequences of subtilases are known; most are derived from he

B

n

Fig.

1.

A:

Schematic representation

of

the secondary structure topology

f

subtilases, with a-he lices shown as cylinders and p-sh eet strands as ar-

rows. Solid lines indicate he conserved regions (scrs) o al l subtilases, and

dashed lines the variable regions (vrs ). Approximate location is indicated

of

the main Ca2+-b inding sites (by Ca l and CaZ), catalytic triad residues

D32,H64,

nd S221 (by

*)

and substrate-binding region (between strands

e1 and em ). B: Ribbon-plot representation

of

the secondary and tertiary

structure

of

subtilisin

(PDB

ode

2SNI),

made with MOLSCRET (Krau-

lis, 1991).Side chains of the catalytic residues are shown in ball-and-stick

representation.

corresponding geneor cDNA sequences. We caution that in many

cases it has not been established whether these genes encode func-

tional proteinsor whether the encoded protein is actually a prote-

ase. Examplesof the latter are the outer-membrane antigenhssal

of

Pasteurella haemolytica

(Lo et al., 1991), and the anti-freeze

protein af70 of Picea abies (EMBL D86598), which were not

described as proteases by the authors.

Themajority of the subtilases are synthesized as pre-pro-

enzymes, subsequently translocated over cell membrane via the

pre-peptide (or signal peptide), and finally activatedy cleavage of

the pro-peptide. A detailed comparison of the pre-pro sequences

and the putative processing sites f these subtilases has identified

two main types of pro-peptide (Siezen et

al.,

1995b). However,

there

are

numerous exceptions n which the pro-peptides appear o

be completely unrelatedor even absent. A small number of subti-

lases is intracellular (Table 1).

Table 1 shows that the (putative) mature enzymes range in size

from 266 o 1775 residues. The catalytic domain or module s

defined as the segment with sequence homology to subtilisins; it is

always located at the N-terminal end of the amino acid sequence

directly after the pre-pro region.This review is focussed

only

on

the catalytic domains.

Alignment of primary sequences

The multiple sequence alignment f the catalytic domains of over

120 subtilases is shownn Figure 2. Additional variants with 10%


3/23

Subtilases

a

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DKVQAQGFKG I9N..VKVAVLDTGIQ sHpDL~ . . . . . . ~ ~ ~ ~ ~ ~ ~

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Q T V ~ ~ ~ ~ p W G I N R ~ Q " ~ . ~ ~ ~ ~ ~ ~ " . ~ ~ ~ ~ ~ R p I A Q S R G ~ T ~ ~ G ~ . v R


4/23

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R.J. Siezen and

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- -NYD LI SCDVNGNDLDPMPRY~-~-...As NEN~G=cACEVAAAA---....

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- - NYDPI ( I I SYDYNGNDGDpMPHC ...L T O s ~ G= CA G E V ~ T A--....~ K C A . ~ G I A Y ~ - - A R V G C V ~ L D - - - - - G D V T D V V E A K S L G L N S Q ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ H ~ D ~ Y S A S W G P D D D ~

..

~ o p K ~ s y ~ v N mo ~ ~p Q p n y...... I I NS~G=CAG~VAAI A

......

~ ~ ~ A . v ~ ~ A F H . . A G I G G V ~ L D ~ ~ ~ ~ ~ G D V ~ A V E A R S L S L N S Q ~ ~ ~ ~ ~ ~ ~ ~ - ~ - - - - - - - Y ~ D l Y S A S W G P D

~ . N ~ ~ ~ R ~ ~ ~ ~ V ~ ~ ~ ~ ~ ~ ~ R ~ . . . . . . . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ G ~ V ~ ~ F . . . . . . . ~ ~ L ~ I . ~ ~ I A Y N . . A N I G G ~ ~ L D ~ . ~ ~ ~ G D V T D A V E A A S V G ~ N A D ~ ~ - - - - - - ~ - - - -

..

YDPYI \ SYDLNDHDNDPM~R~

...... ASNE*G~CAGE SAEA ..~...~-~ ~ ~ ~ ~ I A p D ~ ~ ~ ~ I ~ ~ ~ ~ ~ L D ~ ~ ~ ~ ~ ~ ~ V Y ~ A ~ ~ A A S L S F ~ ~ - ~ ~ ~ ~ ~ ~ ~

.. YDEI ( ASYD NGHD~DP~PRY...... y~E~G=c AGVV- QA.......

v ~ ~ . v ~ V A Y N . . AR I G G V ~ L D ~ ~ ~ ~ -G D V ~ S V E A Q S L G LN S Q - - - - - - - - - -- - - ~ ~ ~ - H I H I Y SA T W G P D D D " " - " " "G R F J D G P A T L A

: : ~ : ~ ~ : : ~ : ~ ; i ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ : : : ~ : : ~ ~ N D ~ ~ ~ ~ ~ : ~ ~ ~ ~ : ~ : ~ ~ ~ ~ : ~ ~ ~ ~ ~ : ~

: ~ ~ ~ ~ ~ a : ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ g ~ ~ ~ : ~ ~ : : ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ : : ~ : : ~ : : : : : ~ ~ ~ ~ ~ : ~ ~

: : ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ : : : : ~ : : : : : ~ ~ ~ ~ ~ ~ ~ ~ : ~ ~ ~ : : ~

: : ~ F ~ ~ ~ ~ ~ : ~ ~ ~ ~ ~ ~ ~ ~ ~ : : : : : : ~ : : : ~ ~ ~ ~ ~ ~ ~ ~ : ~ ~ ~

~~N~D~EASF DI NGNDSOPTP Q~~~~. . . .N~DN*G=cAGEVAAVA ..... ~ ~ ~ ~ ~ ~ . ~ ~ v A y N ~ ~ A s I G G v R M L D ~ ~ ~ ~ ~ G K ~ N D ~ E A Q A L S L N P S ~ ~ ~ ~ ~ ~ ~ ~ - ~

~~N~DPL AST DI NDHDDD~TP Q~ ~ ~ ~ . ~ . .GDN* G~=A~EVAALA .......

~ ~ ~ . ~ ~ v A F K ~ ~ A K ~ ~ ~ v ~ ~ L D ~ ~ ~ ~ ~ G A V S D S V E A A S L ~ ~ N Q D - . . ~ ~ ~ ~ ~

KTFDGPGPLA

~ ~ N ~ D Q T A S I V L N D N D N D ~ ~ ~ R ~ ~ ~ ~ ~ ~ ~ ~ D ~ D A D N ~ ~ ~ = ~ A G E A A A I A

...... ~ ~ c ~ . ~ ~ v A y N ~ ~ A K l G G v R M L D ~ ~ ~ ~ ~ G Q A T D A L E A S A L G F R G D

. . . ~ ~ ~ . - -

~ ~ ~ ~ ~ ~ " I D l Y l ~ C W G P K D D ~ - - - ~ ~ ~ ~ ~ ~ ~ G K

~ ~ N ~ S ~ ~ G S ~ D L N S N D ~ D ~ ~ P H P - - " - - D V E N G ~ ~ ~ ~ A G E ~ A A V P - - - . ~ ~ ~ ~ ~ F ~ A ~ ~ G V A Y G ~ - S R ~ A C I R V L D ~ - ~ ~ ~ G P L T D S M E A V A F N ~ Y Q ~ ~ ~ ~ ~ ~

~~SCKI APRD TRKRI FPTP .~..~......- ~ G T A C A G V A C G ~ ~ ~ ~ ~ ~ ~ ~ . N G ~ G * . S G V A ? G. K = ~~ I ~ F v......L G S Q D E A D S ~ " ~ A ~ Q ~ - - - - ~ - - . - ~ ~ ~ ~ ~ ~ ~ C A D V I S C S W G P P D G ~ - T W W D D R D P L H K Q K V P

rSYAVVSESWGCVDD-----------GAAFCDTTGNF

~~WRP ~CSKWVTGCS DP~p ~ ~ ~ ~ ~ ~ . T ~ D ~ ~ ~ ~ . .V ~G I I A A V~ ~ ~ ~ ~ ~ ~ D N ~ I ~ ~ . L G V A ~ R . . ~ Q L Q ~ ~ N ~ ~ D ~ . . . N I Q Q L Q K D ~ L Y A L C Q R R ~ ~ ~ Q P G - - - . ~ - ~ ~ ~ ~ ~ ~ L Q P E L R M S L V D P E G ~ - - - ~

~ ~ V N C V A C K P D T A D C A W R P S ~ ~ ~ ~ ~ - - - - - I \ I E S P ~ G ~ ~ G E I A A A K ~ - ~ ~ - - - - N G V G ~ - T C V A ~ G - - ~ K V A ~ I K V S N P - - - D G F F Y T E A ~ C G F M W A A E H - - ~ - ~ - ~ - ~ ~ ~ ~ ~ ~ ~ - C ~ D V ~ S Y Y T

: ~ ~ ~ , " ~ ~ ~ ~ ~ , " ~ ~ ~ ~ ~ ~ ~

: ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

y F ? r . , ~ ~ ~ y ~ F ~ ~ ~ ~ ~ ~ ~ : : : : . : : : : ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ : : : : ~ ~ ~ ~ ~ ~

~ ~ I P Y ~ K G D A F R y D G T p S Y D S D " - - - - - - - - C T L G S ~ G ~ v A A S P P A A E ~ ~ ~ - - - - - D G G ~ . H G V A F N ~ ~ A Q I l S A E N G D P ~ 6 ] I L ~ N D ~ A V Y Q A G W D A L V A S ~ ~ ~ ~ ~ ~ - ~ - - - - - - - ~ G A R I ~ ~ S W G I G ~ T ~ ~ D ~ Q K

~ - ~ Q W L G S T N L N I \ H T G I L P I T Y V ~ N V P ~ ~ ~ D S S S G E G ~ A G F J G G T G A - - - - - - M S G G K Y ~ E G V A P G - - E N L ~ G Y G S G A . . . . . ~ V V A M L D T L G G F D Y A L ~ Q Q E Y ~ ~ ~ - ~ ~ - - - - - - N I R I l ~ S W G A T S D " - - - - - ~ ~ ~ ~ A G T

- - V Q N V L G S T N L Q G I T G I L P I T Y T ~ N V P ~ ~ ~ D ~ S ~ G ~ A G ~ G G T G A - - - - - ~ M S G G K Y - ~ G A A P G - - A D L I G Y G ~ G G . . . . . ~ A L F ~ L D G ~ G G F D Y A ~ ~ ~ E Y ~ ~ ~ - - - - - - - - - D ~ R V ~ ~ S W G S S

~~NEP ENEMNWYDAVAGEASP......... Y D D ~ ~ G ~ ~ G T M V G S E - - - - - - - P D G ~ Q . l G V A P G - - A K ~ l A V ~ A F S E - - - - D G G T D ~ I L E A G E W V L A P ~ A E G ~ H P E M " - - - A P D V ~ S W G C G S G " " " - ~ ~ ~ ~ ~ ~ "

~~NFGQYKGYDFVDNDYDPI ( ET... TGDpRGEA~nG- ~~~AANGTl

...........

GVAPD~~ATLLAY RVLGP.. G 5 G T T E W I A G V E R A V Q D - - - - - ~ ~ - ~ ~ - - - - - - G A D V M N L S L G N S L N " - - - - - ~ ~ ~ ~ ~ ~ ~ " N P D

W V N D K V A Y Y H D y S I ( D G K T " - - A V D Q E B G T W S G I L S G N A P S E T ~ - - K E P Y R L . E G A M P E - - A Q L L L M R V E I V N - - G L A D Y ~ Y A Q A I R D A V ~ - - - - - - - - - - ~ ~ ~ ~ ~ - G A K V I N E I S F G N A A L - ~ ~ ~ " " " - ~ ~ A Y A N L P D E T

S C N G K I V G A Q Y F R H G A I A V ~ E ~ - N R T R D Y R S P F D ~ G E G S ~ T A S T ~ G N ~ ~ A ~ ~ ~ N G Y N F G Y A S G M A P G - - A W I A ~ Y ~ L ~ ~ - - - - F G G ~ S D V V A A Y D ~ ~ E ~ ~ ~ ~ ~ ~ ~ - - - - - - - - - - G V D I I S L S V

H C N S ~ L I C I R Y F ~ C I H A A I P - N A T F S M N S R R D T L G E G ~ T A ~ T ~ ~ N ~ N G A S ~ F G Y G K G T A R G I A P ~ - - R R ~ ~ ~ ~ ~ ~ T ~ P - ~ ~ ~ E G R Y T S ~ V L ~ G ~ ~ ~ I A D ~ ~ - ~ ~ - - ~ - - - - - - - ~ G V D V I

R C N R K I I G A R S Y H I G R P I S P G - - - - - - D ~ G P ~ D ~ G E G ~ T ~ S T ~ G G L V ~ ~ ~ L Y G L G L G ~ A R G G V P L - - ~ R I A A Y K V C W N . ~ ~ ~ D G C S D ~ I L A A Y D D A I A D ~ ~ ~ - - - - ~ - - - ~ ~ ~ ~ ~ G V D I I S L S V

K C ~ K L I G A R S Y Q L G H C . ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - S P I D D D G ~ G ~ ~ A S T ~ G A F V N G ~ F G N ~ G T A A G V A P F - - A H I A V Y K V C N S ~ ~ - - D G C ~ ~ V L ~ M D ~ I D D - - - - - - - - ~ ~ ~ ~ ~ - ~ - G V

L C N R K L I G A R F F R R G Y E S M G p ~ D E S K E S R S ~ ~ D D G E G ~ T 5 S T A A G S V V E G A ~ L L ~ Y A ~ G T A R C M L - - ~ - H A L A V Y K V C ~ L - - - - G G C F S S D l L ~ l D ~ l A D - ~ ~ ~ ~ " ~ " " " " W W L S U S L G G G M S ~ ~ ~ " "

KNVKERRI WRTL

.............

DDG~GEGT~VAGVI ASMRE~

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N C N R I ( I I C R R Y ~ S ~ ~ E D D D L K ~ ~ ~ I W P E S R T ~ ) Y Q G ~ C ~ Y T ~ T A A ~ S F ~ N ~ N G L ~ ~ ~ ~ ~ G ~ ~ A S S S ~ ~ A ~ ~ V C G L - - - ~ ~ G ~ P G ~ Q ~ L A A F D D A ~ ~ ~ - - - - ~ ~ ~ -

.~~~

~~~~ . - ~ ~ ~ f y ' : ~ ~ ~ ~ , , I ~ ~ ~ ~ : : ~ ~ ~ : ~ ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ : ~ ~ ~ ~ ~ ~ : : : : ~

~ : ~ ~ : : ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ : : : ~ : ~ ~ ~ ~ ~ ~ : ~ :

~ ~ ~ ~

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D V I A ~ D N G T ~ ~ ~ ~ ~ ~ N G ~ T . ~ ~ ~ ~ ~ ~ S D F H G ~ G T S V ~ ~ I A S R G R V L Y D L Y G D G ~ L ( ~ ~ G V ~ P G - - A K I A G G D A W L L - - - C N I L V L E A N L A G F N I V T E E E D G W Y L S L D P F G P H - ~ D I ~ S N S W G S ~ Y

~~~

~~~~

Fig. 2. Continues.


5/23

Subtilases

140

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Q N A H R N F y Q Q " " . G H L L ~ ~ c N ~ c ~ ~ . ~ . . . . . . . . . ~ . ~ . . . ~ . . ~ . c ~ ~ y p ~ s y ~ ~ ~ ~ . ~ ~ ~ ~ " ~ " ~ ~ . . . . . . . ~ ~ . . . . . . . . s ~ ~ ~ ~ ~ . . . . . ~ Q ~ ~ ~ Q ~ ~ ~ ~ ~ ~ ~ . . . . . . ~ E~~

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- - ~~~~~

T N A V D Y A Y D K - ~ ~ ~ - G Y L I I ~ A I \ G I ( S G P K P G - - - - - - - - - - - - - ~ ~ ~ ~ - - - S I ~ Y ~ ~ A L V - - - - N ~ ~ ~ ~ A ~ ~ ~ N ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - ~ I Q ~ ~ T ~ ~ ~ ~ ~ F ~ ~ ~ ~ ~ S S ~ ~~~~~

E A K ~ ~ S . Q I L W ~ - ~ ~ c ~ ~ D~ ~ ~ ~ ~ . . . . . . . ~ . . . . . . ~ ~ ~ ~ ~ ~ ~ ~ . ~ . .V I S V G A I N F ~ ~ ~ ~ ~ ~ ~ ~ ~ . . . . . . . .~ A S E F ~ ~ ~ ~ ~ S N S ~ E~ ~ ~ ~ ~ ~ ~ ~ V D L V A p G E . . . . . .

.

~ ~~~

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.

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Fig. 2.

Continues.


6/23

506 R.J. Siezen

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~~N I L S T Y I GS - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - ~ - - - - - - - O D I T A T L - S G T S H I S P R Y I C L L T Y F L S L Q ~ C S D S E F F E L G Q ~ ~ ~ D S L T P Q Q L K K ~ ~ ~ ~ ~ ~ T ~ ~ ~ ~ ~ - - - -5 1

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- - O I I S A S Y Q S - - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D S G T L V Y ~ S G T S M A C P H V A G L A ~ Y Y L ~ l ~ - - - - - - - - - - - - - - E V L T P A Q V E A L I T E S N T G V L P T ~

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- - EI ESLSHLN~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - ~ Y N D T L I L - S G T S M S T P l V T G V ~ ~ L L ~ K C . . . - - ~ - - - - ~ - ~ - - - - I E P E M I A Q E I E Y L S T R ~ F H R R T L - F F I ( P5 1

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~ - E I I T T I G T D A I W I D F Q F I E N V P R G F I l n - I G T S L I T G L F ~ I ~ - - - - - - - - - - - - - - - - - -- - - S L QR F KS A NF Y

K Q S V L S T S S ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - ~ - ~ ~ - - - - N G R Y I Y Q - S G T S L R ~ P I Y S G I \ L R L E I D I ( Y Q - - ~ ~ - - ~ ~ ~ - - -L ~ O Q P E T A I E L F r K r c l E K E r Y H D R X B Y G N C r L D V Y K L L K E

KDWLFTTAN ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ T G W Y Q Y V ~ Y C N S F A T P K Y S G A L ~ L ~ ~ D K ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

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- - - ~ ~ ~ - ~ ~ ~ ~ - ~ ~ ~ ~ ~ M I C T A S H ~ S ~ T S A A A P E A A C Y P R L R L E A ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ N L T W R D M Q ~ L N L T S K ~ N ~ ~ ~ D ~ - - - ~ ~ - - - - N ~

~ ~ E " L A l D K ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ " " " " " " " . Q S E I T I Q . S G T S F I \ T P ~ " ~ ~ " ~ ~ L y l E D C E " ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ S I D L D F L R S I ( S E D L G " " " " ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

E T C V A T mL y - ~ ~ ~ ~~............... R C T R S H - S G T S A A R P E A A G \ I F R L A L A L ~ A N P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ S L T W R D L Q H L N L T ~ ~ ~ N ~ ~ ~ D ~ ~ C ~ F I I ~ l N C S H F E U ~ N G V G L E Y I D M ( L F G

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N C T L ~ R - S G T S ~ A P E A A C Y F R L A L A L Q A N P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ N L T W R D ~ Q " L T " L T ~ ~ ~ N ~ ~ ~ ~ ~ V H E - - - - - - - ~ - ~ ~ - W ~ N G V G L E F I D M (

E A G V A T T D L Y ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ G N C T L R H - S G T S A A A P ~ ~ ~ ~ ~ F ~ L ~ L E A N L - - - - - - - - - - ~ - - - - G L T W R D M Q H L T ~ L T ~ K ~ N Q L H D E V H Q - ~ - ~ ~ ~

D P R I T S A D L H ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - N E C T Q T H - T G T S A S A P L A A ~ I F A L A L E Q N P - ~ ~ ~ ~ - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ L T W R D L Q H I V V W T S E F D P L A ~ G - - - - - - - - - - - - -

E C R V T S A D L H ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - G K C R I S R - ~ ~ T ~ ~ A A ~ ~ ~ A ~ L ~ A L L L E S N P ~ - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ N I T W R D A Q ~ ~ ~ A H T S R M E

D Q R I T S A D L H ~ ~ ~ ~ ~ ~ ~ - ~ - ~ ~ - - - - - - - - - - - - - - N D C T E T H - T G T S A S A P L A A G I F A L A L E A N P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ N L T W R D M ~ H L ~ ~ ~ T S E Y D P L A M I P G ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - - W

D Q K I S S ~ L H ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - - - - - - - - - - - - - - - H E C T D S H - T C ~ S A A A P L A A G ~ L A L A L E A N P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ N L T W R D V Q ~ L I V W T S E Y D P L S S ~ G ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - -

D K K I I ~ D L R ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - - - - - - - - - - - - - - Q R C ~ I D M ( ~ T C T S A S A P M A A G I I A L A L E A N P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ F L T W R D V Q H V I V R T S R A G H L N A ~ ~ - ~ ~ ~ ~ - ~ - - ~

E R K I V ~ D L R ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - - - - - - - - - - - - - - Q R C m C H - T G T B V S R P M V A C I I A L A L E A N S ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Q L T W R D V Q H L L ~ K T S R P A H L K A S D - - - - - - - - - - - - - - ~ ~ ~ N ~ A ~

E K Q V I ~ L H ~ ~ - - - - - - - - - - - - - ~ - - ~ - - - - ~ ~ - H S C T S S H T C T s R S R P L A A G I A A L V L E A N P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~ ~ N L T W R D L Q ~ I V V R T A K ~ G N L K D P T ~ ~ ~ ~ ~ ~ ~ ~~ ~ ~ ~ W S ~ N G Y G R R V S H S F G Y G L M D A A H V I L A O

E K Q V V T ~ L H - - - - - - - - - - - - H S C N S H T C T B R S R P L R R G I A A L V L Q S N Q ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - ~ - ~ N L T W R D L Q ~ I V V R T A K P A N L K D P S ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ S R N G V ~ R R V S H S

E R E I I T S D L H - ~ - - - - - - - - - - - - - - ~ - ~ - ~ - ~ ~ ~ ~ H S C T T Q H ~ T G T S A S A P L A A G I C A L A L E A N K ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Q L T W R D M Q H I V V R T A R L A N L Q S S D ~ ~ ~ ~ ~ ~ ~ ~ - - - - - - ~ ~ T N ~

E K ~ I L T T D L H ~ ~ ~ ~ ~ ~ ~ ~ - - - - - - - - - - - - - - - - - - H A C ~ H - T G T S A S A P L A A G I V A L A L E A N P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ N L T W R D L Q H ~ V I R T A K P I N L ~ G D - - - - - - - - ~ - - - - - W T T N G V G R

E K Q I V T ~ L H ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - - - - - - - - - - - - - ~ ~ ~ ~ ~ ~ - ~ ~ T ~ A S A P I V ~ ~ L L A L A L E A N P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ S L T W R D L Q ~ I I ~ E T A K ~ D ~ L ~ ~ D ~ - - - -

E R Q I A T T D L R ~ ~ ~ ~ - ~ ~ ~ ~ - - - - - - - - - - - - - - - - - Q R C T T I 1 I - T G T S l S A P L A A ~ I ~ A L ~ L E A ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D L T W R D V Q Y I T L M T S R S D P I ~ D G Q - - - - - - - - - - - ~ - - W I V N G V C R

O P Q I V ~ L H - - ~ - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ H Q C T D K H T G T S A S R P L A A G M I A L ~ L E A N P - - - - - - - - - - - - - - - L L T W R O L Q H L V V R A S R P A Q L Q A E D ~ ~

E K C I A S T D L H ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~ ~ - - - - - - - - - - - - E K C T ~ - T G T S I \ S I ~ ~ ~ ~ ~ ~ ~ E - - - - - - - - - - ~ ~ - ~ W V T N C V G R Q V S L R Y G Y

E I ( Q I V T T D L R - - - - - - - - - - - - - - - ~ ~ ~ ~ - ~ ~ ~ ~ ~ ~ Q K C ~ S H ~ T C T S A S A P L A A G I l A L A L E A N K ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~ - N L T ~ R D M Q H L V V Q T S N P A G L N A N D ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

E K Q I V T T D L R - - - - - - - - - - - - - - - " - Q I [ C T E S H T C T E S H ~ T G T S A S A P L A A G I I A L T L E A N K - ~ ~ ~ - ~ ~ ~ ~ ~ - - - - - N L T W R D M Q H L V V Q T S K ~ A H L N A ~ ~ ~ ~ - ~ ~ ~ ~ ~ - ~ ~ - - W A T N G V G R K V S H S Y G Y G L L D

Q P A I V N D V P - - - - - - - - - - - - - - - - - - - - ~ ~ ~ - ~ ~ G G C ~ K H ~ T G T S A S A P L A A C I I A L A L E A N P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ E L T W R D M Q H L V L R T A N ~ K P L E ~ G - - - - - - - - - - - - - W S R N G V G R M V S N

D K S V A N D H D G S L R P D - - - - - - - - " - H I C ? 1 I E H T C T E R S A P L A A G I C A L A L E A N P ~ ~ ~ ~ ~ ~ ~ ~ - - ~ ~ - ~ ~ E L T W R D M Q Y L V V Y T S R P A P L E ~ E N C ~ ~ ~ - ~ ~ ~ - - - - - - ~ T L N ~ V K R K Y S N K F G Y G L M D A G A

E N ~ H Y ~ L Y - - - - - - - - - - - - - - - - - - - - - - - - ~ - H ~ T E E F ~ K G T S A S A P L A A G I ~ A L T L E A N P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ L L T ~ R D V Q A L I V H T A Q I T S P V D E ~ - - - - - - - - - - - - - - W ~ R N C R C F H F ~

L R S I V T T D W D L Q K G - - - - ~ ~ - - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ T ~ C T E C H ~ T ~ T S A A A P L A A ~ M I A L M L Q V ~ P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - C L T W R D V Q H I I V F T A T R Y E D R R A E - ~ ~ ~ ~ ~ ~ - - - -

- - Y I Y G T D I N A I D D K S R R - - - ~ ~ ~ - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ P R C Q N Q H ~ G G T S A R A P L A A G V F A L A L S V R P - - ~ ~ - ~ ~ ~ - - - ~ - - - D L T W R D M Q Y L A L Y S A V E I N S N D D G ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - - ~ Q D T A S G Q

- - Y I I T T D L D - - - ~ - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - E K C S K I 1 ( - G C T s l U \ R P L A A G I Y T L V L E R N P - - - - - - - - - - - - - - - N L T W R D V Q Y L S I L S S E E I N P H D G K ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - W Q D T ~ G

- - Y ~ H S S D I N ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~ - - ~ ~ ~ - - - G R C S N S H - G G T S ~ A P L A A G ~ Y T L ~ L E A N ~ - - - - - - - - - - - - - - - N L ~ W R O Y Q Y L S I L S A V G L E I W A D G D ~ ~ ~

~ ~ S I L ~ P E ~ ~ - - - - - - - - - - - - - - - - - - - - - - - - G T C T R S H - G G T S A A A P L A S A ~ Y A L A L S I R P - - - - - - - - - - - - - ~ ~ D L S W R D I Q H ~ ~ Y S A S P F D S P S Q N A E - - ~ - ~ - - ~ ~ ~ ~ ~ U Q K T P A G F Q F S H H F G F G K L D

~ ~ P N E ? V U Y D ~ - - - - - - - - - - - - - - - - - - - - - - - - - G K C G F I P - S S S S A R P P I L G ~ L L A L I R A H P - - - - - - - - - - - - - - ~ T L T L ~ I Q R I L ~ R A A ~ ~ V ~ T ~ ~ G R G W ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ W L M l V ~ R ~ ~ R N F

T P G I W T R D R T G V - V G Y N S G N L G D Q A - - - - - - - - - G N Y R I ~ ~ - ~ ~ T S ~ A C P ~ - ~ " ~ ~ L I L S ~ N ~ - - - - - - - - ~ - - - - - - ~ ~ ~ ~ D ~ " ~ D I I K R ~ C D R I D P V G G - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - ~ N A E

81

A P A ~ V T ~ L P G C D U G Y M l V D D P S T N R L H M I P Q L D l S C D Y N G ~ ~ ~ - - - - - - - - - - - - - - ~ ~ D L S Y R D L R D L L I ~ N I T R L D A N ~ P V Q I N Y I 9 ) V T G L E C W E R N A A G L W Y S P S Y G F G L V D V N K T Q P C S I I l

- - L I L G T L P - - - - - - - - - - - - - - - - - - - - - - - - - - - G G K ~ G Y M - A G T S M A S P H V A G V A A L l K S ~ P - - - - - - - - - - - - - - - H A S P A M V K A L L Y A ~ A D A T A C T K P Y D l D G D G K V D A V - - - - - - ~ E ~ P K ~ ~ C F Y G ~ G M A D A L D A V T W

- - D I Y S T Y P - - - - - - - - - - - - - - - - - - - - - - - - - G C G Q C T Y P - - - - - - - - - - - - ~ ~ ~ D ~ T P A Q I ~ T R I E ~ T A E R S V N G ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - ~ - - - - - - - - - H D D F V G

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~ ~ D I Y S N C R L E S ~ G C A V M ( E A Y N K G E L S L ~ ~ - ~ ~ N P G Y G N K - S G T S M A A P H V T G V A A V L M Q R F P ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Y ~ S A D Q I S A V I K ~ A T D L G V A - - - - - - - - " G I

- ~ R V Y S S I I E G T S V E N L - - - - - - - - - - - - - - - - - - - T T G Y A K Y - S G T S M A A P H V A G S V A V L M E R F P - - ~ ~ - - - - ~ - - - - - - Y L N G A Q V A E V L K T T A ~ M G A P ~ ~ ~ ~ - - - - - - - - - - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ G I D A L Y G W G M

- - I ( I Y S N R N G S D P ~ - - - - - ~ - ~ - ~ ~ - ~ - - - - - - ~ S D Y G N K - N G T S M A T P H V T G A V A ~ L L Q R F P - - - - - - - - - - - - - - - ~ ~ S S A Q I A D V L K T T A ~ M G A P ~ ~ ~ ~ ~ - - - - - - - - - - ~ - - - ~ ~ ~ ~ ~ ~ C I D A L Y G W G I I I

- - L I G V A D E H K K P - - - - - - - - - - - - - - - - - - - - - - - Q Y G L T K E - ~ T S F S A P A I T A S L A V L K E ~ ~ D - - - - - - - - - - - - - - - ~ ~ T A T Q I R D T L L T T A ~ L G E K - - - - - - - - - - - - ~ - ~ ~ ~ ~ ~ ~ ~ ~

- - O I Y S A R Y F T P L S A L S A Q I L E Y I S P R H - - - - - - - L P Y Y T T F - S G T S M A A P H V A G I l A L M L E ~ ~ - - - - - ~ - - - - - - - - - ~ ~ ~ ~ L E ~ K E I L E G T A l P M E G Y - - - - - - - - - - - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ A l W E T

~ - D I Y S ~ R V L A P L S A L M E I A ~ L l ~ P Q H - ~ - ~ ~ ~ ~ L P Y Y T ~ ~ S G T S M A T P ~ A G l V A L M L E A D P - - - - - - - - - - - - - - - T ~ ~ P D Q V K E I ~ Q H T A ~ P G Y ~ ~ ~ ~ ~ - - - - - - - - - - - - - ~ ~ ~ ~ ~ ~ ~ ~ E A

~ ~ N I R S S V P ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~ ~ ~ ~ ~ - - - - - - - - - - ~ ~ G Q T Y E D G ~ D G T S M A G P ~ V S A V A A L L K Q ~ A - - - - - - - - - ~ - - - - - S L S V D E M E D I L T S T A E P L T D S T ~ ~ ~ - - - - - - - - - - ~ ~ ~ - ~ ~ - ~ ~ P D S

~ ~ N I V S T I P ~ P D H ~ ~ ~ ~ - - - ~ - - - - - - - - - - - - P Y C Y C S I ( Q C T S M A S P H ~ A G A V A V I K Q A K P ~ - ~ ~ ~ - ~ ~ ~ - - - - - - K W S V E Q 1 K A A ~ M ~ A V T L K D S D ~ ~ ~ ~ ~ ~ - - - - - - - - ~ ~ ~ ~ ~ ~ ~

~ ~ D I L S S V A - ~ ~ - - ~ ~ - - - ~ - - - - - - - - - - - - - - - - M I K Y A K L ~ S G T S M S A P L V A G I M G L L Q K Q Y E ~ ~ ~ ~ ~ ~ ~ T Q Y P D M T P S E R L D L A K K ~ L M S S A T A L Y D E D ~ ~ ~ ~ ~ ~ ~ ~ - - - - - - - - - - ~ ~ E ~ Y F

~ ~ N I W S T Q N ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - ~ ~ ~ M I C Y ~ ~ S C T S M A S P ~ l A ~ ~ Q ~ ~ ~ K Q A L M I K M I ~ ~ Y A ~ ~ ~ Q ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ? V

- - ~ l Y S L I \ ~ - - - - - - - - - ~ - ~ ~ - ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~ ~ - D N K Y Q Q M ~ S G T S M A S P F ~ A ~ S E A L ~ L Q G ~ ~ - - - - - - - ~ Q ~ ~ N ~ ~ ~ ~ ~ ~ ~ Q F ~ ~ ~ A ~ ~

- - S I W R A W S S N S T E - - - - - - - - - - - - - - - - - - - - - - G E N F A L ~ - S ~ T S M A T P ~ A G ~ A ~ ~ ~ K Q ~ H P - - - - - - - - - - - - - - - N W S P ~ l A S A l M ~ A Q ~ D ~ ~ ~ L L - ~ ~ ~ ~ ~ ~ ~ ~ ~ A Q Q A T ~ P S T A T

~ ~ L Y L A S W I P N E A T A Q I C R i Y Y L - - - - - - - - - - - - - ~ ~ H ~ ~ ~ - ~ ~ T S M A ~ P H A ~ G V ~ A L L ~ A H P ~ ~ ~ - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ E W S P A R l ~ ~ ~ ~ ~ ~ ~ N ~ ~ ~ N T L N P l ~ ~ ~ ~ ~

~ ~ N I L A A W P T S V D D N ~ - - - - - - - - - - - - - - - - - - K S T F N I I - S G T S M S C P H L S G V R A L L ~ S ~ P ~ ~ ~ ~ ~ ~ ~ ~ ~ - - ~ ~ ~ - D ~ ~ P ~ ~ K S ~ M ~ R D T L N L A N S P I - - - - - - - - - - - - - - L D E R L L P A D ~ Y A I

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- ~ N I I A A W N P P N Q S D E D T W S E H T - - - - - - - - - - - - P S T F M L L ~ ~ ~ - ~ - - -- - - - T N ~ S D ~ P G T P F D F G A G W N P I C R L P P C o

~ ~ N ~ L A A U T G A R G P T G L A S D S R - - - - - - - - - - - - - - R V E F N I I ~ S G T S M S C P H V S G L A A L L K S V H P - - - - - - - - - - - - - - - ~ ~ ~ P ~ ~ R S A L M ~ A Y K T Y K D G K ~ ~ - - - - - ~ ~ ~ ~ ~ ~ - - L D ~ A T G K P S T P F D H G A G H V S P T T A

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- - Y ~ ? . ~ ~ ~ ~ N ~ E N S T o Q C G D G S L P N - - - - - - - - - - - R N ~ ~ ~ ~ ~ - ~ ~ T S ~ A T P L A T A A T T l L R Q Y L V D G Y F P T G E S V E E N K L ~ P ~ ~ ~ ~ ~ ~ ~ L ~ l M I A Q L L N G T Y F W S A S S - - ~ - ~ ~ T N P S N A ~ F E Q l N C A N

- - Y I T S ~ S N G ~ ~ Q C GD G S L P N - - - - - - - - - - -~ A L L A l - S G T S M A T S F AA A A ~ l L R Q Y L V D G Y Y P TG S l V E S ~ L Q P T G S L L K AL M l M I A Q L L N G T F Q L lT S S S l ~ ~ ~ - T Y P S N Q V FE N F A G A S L V ~ W G A I ~ S NW L H V l l O ~ 2 l

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.. FAGYPQYC......................... R Q ~ M - ~ . N ~ T S ~ S ~ ~ N - G - A C M L ~ G L K. . . . . . . . . .Q ~TPY? VRMALE~AYMLP I . . . ~ ~ ~ ~ ~ ~ ~ - - - - - - - - - - -~ ~ ~ E S F S Q G ~ U l K l A T A Y E K L i ~ l 3 l

. F E U A S ~ T I D C R G Y ~ ~ ~ . . . . . . . . . . . . . . ~ . ~ ~ ~ A Q p D V F - ~ ~ T ~ ~ A T P y T S ~ T ~ A L ~ ~ Q A Y K E - - - - - ~ ~ ~ - ~ V y ~ T p D p ~ T A ~ ~ ~ L K S S A K D I W Y ~ ~ - - - - - - - - - ~ - - ~ ~ ~ ~ ~ ~ - - - - - - ~ P A F S Q C S G R M A L K A R

.. YSSLPMW

.........................

1 G ~ ~ F M . s G T s M ~ T p ~ V S G ~ A L L I s G p K.......... ~ ~ ~ y ~ p D ~ ~ ~ ~ v L E s ~ A T ~ L E G D P ~ ~ ~ ~ ~ ~ ~ - - - - - - - - ~ ~ ~ ~ ~ T G Q K Y T

~~HI~.SSLPLWYTV-S ~ ~ ~ ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ ~ . . -~ ~ ~ . ~ ~ ~ ~ ~ A ~ ~ ~ ~ ~ ~ ~ ~ A L ~ I ~ ~ A K ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Q ~ ~ ~ ~ ~ ~ ~ ~ ~ A L ~ L ~ ~ K

I

Fig. 2.

Continued

(see

facing page for

caption).


7/23

Subtilases

sequence difference are not shown in Figure

2

but are listed in

Table 2. Amino acid numbering used throughout this review cor-

responds to that of mature subtilisin BPN (acronym basbpn), our

reference sequence. Residues in inserts relative to this reference

sequence are numbered in square brackets; for instance, residues

inserted between positions 12and 13are numbered 12[+ 1],12[+2],

etc.,

or

13[ -21, 13[- 11 if more appropriate.

The conserved catalytic residues Asp 32, His 64, and Ser221 are

highlighted in Figure 2, as is the oxyanion-hole residue Asn 155.

Conserved core elements (black bars) and secondary structure are

indicated (Siezen et al., 1991,Heringa et al., 1995).This structural

framework can be used for homology modeling of subtilases of

known p rimary structures but unkno wn three-dimensional structures.

In some of the most highly diverged sequences there are regions

with very weak sequence homology, even in the core, which re-

sults in alignments that are not unambiguous. In those cases, al-

ternative alignments to those in Figure 2 may need to be considered.

These regions are found on the surface of the molecule and contain

numerous olvent-exposed residues, allowing orgreater ide-

chain variation. Examples are (a) the exposed regions 43-58 and

182-21 8, which contain structurally conserved P-strands and turns;

and (b) the exposed amphipathic helices 104-1 16, 133-144. and

243-252. In the latter case, the sequence alignm ent of amphipathic

helices is also based on the requirement that at certain positions

non-polar side chains are conserved that point into the interior of

the molecule, while polar residues face outward. When necessary,

correct three-dimensional positioning of Cys residues to form

putative disulfide bonds was used as an aid in proper sequence

alignment.

Sequence homology and family division

In Figure 3, the pairwise sequence identity within the catalytic

dom ains s plotted graphically for all members of the subtilase

superfamily aligned in Figure 2. It is clear that clustering occurs

into groups or families, in which members show higher sequence

identity to each other.

Figure 4 shows the parts of a family tree or cladogram, a mea-

sure of the sequence homology between superfamily members,

constructed from the sequence alignment of the catalytic domains

in Figure 2. In our earlier paper, a less extensive tree identified two

main classes and some subclasses (Siezen et al., 1991).This ex-

panded sequence information now allows a new subdivision into

six families, which are summarized below. The dendrograms in

Figure 4B illustrate the sequence homology within these families

and further subdivision into subgroups (or subfamilies). Many of

these subgro ups are also apparent from the color patterns of se-

quence identity in Figure 3.

507

Subtilisin family

Only found in micro-organisms as yet. Includes mainly enzymes

from Bacillus, with subgroups of true subtilisins (>64 identity),

high-alkaline proteases

(>55%

identity), and intracellular prote-

ases (>37 identity). Numerous minor variants of true subtilisins

and high-alkaline proteases have been identified (Table 2). Long

C-terminal exten sions are rare. Several 3D structures are known

(see Tables 1 and 2).

Thermitase family

Enzym es found only in micro-organisms, including some thermo-

philes (>55% identity) and halop hiles. The c haracteristic N-terminal

sequencewas also ound in severalother Bacillus proteases

(Table 3). Only one 3D structure is known (thermitase).

Proteinase K fam ily

Large family of secreted endopeptidases found only in fungi,

yeasts, and gram-negative bacteria as yet; the bacterial subgroup

has >55 sequence identity. This family is characterized by a

high degree of sequence similarity (>37 identity), only minor

insertions and deletions and the absence of the Ca2+ -bindi ng oop

residues 76-81. Only a few of these enzymes have a significant

C-terminal extension beyond the catalytic domain. One 3D struc-

ture is known (proteinase K).

Lantibioric peptidase family

A small number of highly specialized enzymes for cleavage of

leader peptides from precursors of lantibiotics, a unique group of

post-translationally modified, antimicrobial peptides (Sahl et al.,

1995). Theseendopeptidases have only been found in gram-

positive bacteria, and several are intracellular. Only llnisp has a

C-terminal extension, which acts as a membrane anchor. Charac-

terized by low sequence similarity with each other and other sub-

tilases (Fig. 3), and by numerous insertions/deletions. The most

recently reported protein bspara from Bacillus subtilis is described

as a putative protease required for plasmid stability; we speculate

that it may also play a role in lantibiotic processing.

A few 3D structures have been predicted by homology modeling

(Siezen et a]., 1995a;Booth et al., 1996).

Kexin family

A large group of proprotein convertases (PCs) have been iden-

tified, all involved in activation of peptide hormones, growth fac-

tors, viral proteins, etc. (Barr,

1991;

van de Ven et al.,

1993).

High

specificity is seen for cleavage after dibasic (Lys-Arg or Arg-Arg)

or multiple basic residues. Nearly all are eukaryotic and have high

sequence homology (>40 identity), while two more distant mem-

bers from Aeromonas and Anabaena provide links to other subti-

Fig. 2. ( f ucing page ) Alignment of amino acid sequences of catalytic domains of subtilases. Multiple sequence alignment was initially

performed using the PILEUP program (Devereux et a l., 1984). Nex t, improvements were made manually by taking into accoun t the

structure-based alignm ent (Siezen et al., 1991;Heringa et al., 1995).Inserts were udged to occur m ost likely in turns in external loops.

Fam ilies A to F are indicated on the left. E nzym e acronyms are given in Table 1 . (*) New entries, and (c) corrected entries since S iezen

et al.

(1991).

Residu e numbering at the top corresponds to that o f mature subtilisinBPN (basbpn). Catalytic residues A sp 32, His 64,

and Ser

221

are in bold (hig hlighte d red), as is the o xyanio n-hole residue Asn 155. Green

=

highly conserved residues from Table 4;

yellow = Cys residues. Structurally conserved regions of the coreABC and extended coreAB are shown as solid bars; common

secondary structure elemen ts are show n as: h = helix,

e

= extended p-sh eet, b = bend and t =

p-turn

(see also Fig. I ) . The number

of additional residue s in arge nserts n

the

catalytic domain, and n N- andC-terminal extension s, are show n in brackets.Each

seque nce begins at the mature N-term inus; an N-terminus based on the predicted pro-peptide cleavage site is indicated as().

Resid ues 146-156 of bspara are from a different reading frame than

proposed by the authors.


8/23


9/23

Subtilases 509

k4

E

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w

s.pmvi %

B P % s q g g E g s % ; z g g p gm t -

m 8

NOS %

m

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gGr??zEgggY-

~ i d x E o o x a ~ Z E a ~ ~ ~ ~ a

2 o a m >

s X E E ~ G E E Z S Z E Z

2

N .-

2

e

w m m

+ + + + + + + + + + + + + + + + + e . e . + + + e . + + ++ - + + + + e . + + + +

t - v i - * m - m t 2 - Z m t - B m m

h

8

- m c m o ; z :

o o * m ' D s F i % $ z z g g & g z z

? 2 2 g g g g z s & w g m g % n e ' *vi z * z z w -

% z s ~ s g g ~ ~ g ~ g g z g ~ ~ e .

' =

o m m m

l o , , p ? m m O N N m e N m N

m

ar=i

8 6 Z Z : 3

m m - - o - t - * v i m m m

A C

Q ' z g m t 2 G l o w

m m t - w m - o - -

v

x z g v

e . r n r n 2 m z z l 3 z

- A

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m v i m O v i w e ~ m m - b P - m N ~ O

e r - m ~ m m m t - ~ m m m

q q l o * P - w v i m w m * v i m

w w

v i v i m m ~ m m -

m m m \ g t - w m w

0 0 0

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" -

A - A -' A

e e e e e e e g e e . s g s s g g s r . E L

s s e . ; s g

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3 GLG L G L G L G

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v i N g g

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3

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2 2 $ & w P m m m

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r : 2 C C Q C -

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12/23

512 R.J.

Siezen and

J.A.M. k u n i s s e n

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13/23

Subtilases

0 - m

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;=F m m

mo o

m o p W W P - F :

m m F W

X X N X P E D

E E E ~

Z

2 8 3

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2 %

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g

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$ $ $

g g g m m W m . lz g E %

m u 2 0

W d O

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al

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8 5 6 6 5

8

2

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2

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a a a a

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E Z E E

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P)

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:

3 %

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6 5 5 .5 E

C

g g & E E

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z g $

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E

0 .-

I

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E E E

2 2

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g,

u

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2

2

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s

2 - 8

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s s

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9

2 :

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5

E * =

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3 f %

f %

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14/23

514 R.J. Siezen and J A M . Leunissen

A

A B

C D

E

F

Fig.

.

Pair-wise sequence dentity matrix. Sequences are plotted vertically and horizontally n the same order as in Figure 2; the

incomplete sequenceof hvccvp is not included. Subdivision into families to F is indicated.A color codebar for percentage sequence

identity is shown.

lase families.Asubgroup of yeastenzymes is evident,asare

subgroups ofPC1

( 2 3 %

identity), PC2 (>73% identity), and

furin ( X 5 5 dentity). In catfish herpes virus 1 a related but in-

complete amino acid sequence has been found that is presumedo

have been captured from a host (Rawlings and Barrett, 1994).

Several 3D structures have been predicted bymodeling (see

below).

Pyrolysin family

Heterogeneous group of enzymes of varied origin and low se-

quence conservation (most 37% identity) are distinguished; the former are

of

higher

eukaryotic origin, but onlyhe human and mouse enzymes have ac-

tually been identified biochemically as tripeptidyl peptidases.

Several 3D structures have been predicted by modeling (see

below).

Several other subtilases have been identified or which only the

N-terminal or other partial sequence of the purified enzyme s

available; based on sequence alignment with Figure 2, these sub-

tilases presumably belong to families A,

B,

and C (Table 3).

Conserved residues

Highly conserved residuesare listed in Table 4 and highlighted in

Figure 2. Only the essential catalytic triad residues D32, H64, and

S221 and a single glycine residue (G219)

are

totally conserved n

all sequences.

Four

other glycine residues34,65,83, and 154) are

varied only once or twice; G34 and G154 have main-chain torsion

angles that do not allow for

amino

acid residues with side chains.

At several other positions the variation s limited to two

or

three

residues,whichareusuallystructurallysimilar. In general, the

residues of the two internal helices hCnd hFare the most highly

conserved in

all

subtilases. Three

amino

acid sequences (lslasp,

sepepp, and asaspa) are particularly poorly conserved; although t

seems questionable whether these enzymes are functional, a mu-

tation analysis of the

pepP

gene suggests that

it

indeed encodes a

functional protease (Meyer et al., 1995).

Many more residuesare totally conserved within each of the six

families

A to F,

and hese can

be

used to identify new family

members. In particular, families and C are most conserved, with

a total of 32 and 41 invariant residues, respectively, while family

E

has 63 invariant residues if the

two

more divergent sequences

(asaspa and avprca) are excluded.

Residue N155 (in a conserved segment 152-155), which helps

to stabilize the oxyanion generated

in

the tetrahedral ransition

state (Carter and Wells, 1990), is not fully conserved. The only

accepted substitution here is N155D, as is found in the PC2 sub-

group of the kexin family. The effect of

this

substitution on the


15/23

Subtilases

Families

E T d

1

Lantibiotic

A

peptidases

B

Fig. 4.

Family tree

or

dendrogram analysis of the sub-

tilase superfamily, based on sequence alignment of the

catalytic domains only (Fig.

2).

A: General layout of

the relationship between families A to F.

B:

Detailed

dendrograms of the individual families, in which branch

lengths are in inverse proportion to the degree of se-

quence similarity. Not includedare members with >90%

sequence identity to one of the listed enzymes

(see

Table

2).

Trees were constructed using the neighbor-

joining method of Saitou and Nei (1987), as imple-

mented in the programs NEIGHBOR (Felsenstein,

1993)

and GROWTREE (Devereuxet al.,

1984).

The dis-

tance matrices that were used

as

input for the programs

were calculated using DISTANCES (Devereux et al.,

1984),

PROTDIST (Felsenstein,

1993),

and

HOMOL-

OGIES (Leunissen. unpubl. obs.). Positions containing

gaps were ignored,

as

were the large insertions indi-

cated between brackets in Figure 2. Whenever appro-

priate, the distances were correctedormultiple

substitutions (Jukes and Cantor. 1969; Kimura, 1983).

All

methods used delivered in principle identical to-

pologies, except for the branch lengths; these may vary,

depending upon the method used to calculate the dis-

tances between the proteins, and correcting for multi-

ple substitutions.

subtilisin

true

igh-alkaline

7 1

ntracellular

515

FAMILY

Subtilisin

c

catalytic efficiency of these proteases has been investigated by

protein engineering (Benjannet et al., 1995; Zhou et al., 1995).

Homology modeling

The procedure for homology modeling and protein engineering of

the catalytic domain of subtilases of unknown 3D structure based

on known crystal structures was described in our previous review

(Siezen et al., 1991), and can be applied to any of the enzymes

listed in Tables 1 and 2 .

Modeling should be based on the known crystal structure of the

most related enzyme, and this will be straightforward for members

of the families A-C, because 3D structures are known in each

family. For the families D-F, with

no

known 3D structures, mod-

eling will be less straightforward and can be based on any

known

structure from families A-C or a combination of these. Problems

will arise where large insertions occur, because these are still im-

possible to model reliably. It would be extremely helpful for mod-

Proteinase K

1

antibiotic

peptidase

Kexin

7

7

gram-negative

bacteria

gram-positive

bacteria

3

lant

7

ripeptidasr

2 hermophile

-

Pyrolysin

eling purposes to determine the crystal structure of at least one

member of each of the D-F families, preferably those with large

inserts.

This homology method has since been refined and applied for

modeling and engineering of (a) the cell-envelope proteinase llprtp

of

Lactococcus lactis

(Siezen et al., 1993; Bruinenberg et

al.,

1994a,

1994b); (b) the lantibiotic leader peptidases llnisp of

Lactococcus

lactis

(Van der Meer et

al.,

1994; Siezen et al., 1995a), and efcyla

of Enterococcus faeca lis (Booth et al., 1996); (c) the kexin family

members furin (hsfur: Creemers et al., 1993; Siezen et al., 1994)

and PC2/PC3 (Lipkind et al., 1995); and (d) the heat-stable pro-

teases pfpyro and tsplst of the hyperthermophiles Pyrococcusfu-

riosus and Thermococcus stetteri (W. Voorhorst, A. Warner, W. de

Vos, R. Siezen, in prep.). These studies have provided predictions

and evidence for inserted and disposable loops, disulfide bridges,

&'+-ion binding sites, surface salt bridges and networks, aromatic

surface clusters, and residues involved in enzyme-substrate inter-

actions. Some examples are discussed below.


16/23

516

R.J. Siezen and J.A.M. Leunissen

Table 3 . Incomplete amino acid sequences of subtifuses

Organism Enzyme

Acronym

Residues determined

N-term. Other Family References

BACTERIA

Gram-positive

Bacillus

subrilis

A50

Bacillus sp. (3x6644

Bacillus

sp. Y

Bacillus thuringiensis israelensis

Bacillus thuringiensis finitimus

Bacillus thuringiensis kurstaki

BaciNus cereus

Bacillus intermedius

3-19

Nocardiopsis dassonvillei (prasina)

Gram-negative

Streptomyces rutgersensis

Thermus

Tok3A

1

Vibrio metschnikovii

Cochliobolus carbonum

EUKARYA

Fungi

Agaricus bisporus

Malbranchea suljurea

Ophiostoma piceae

V e r r l c l l l iu ~ ~ h l a m y d o s p o r ~ u m

Scedosporium apiospermum

Intracell. serine protease

Subtilisin GX

Protease BYA

Extracellular serine protease



Extracellular

serine

protease

Alkaline serine protease

Alkaline serine protease

Proteinase D

Caldolysin

Alkaline protease VapK

Extracellular protease


Thermomycolin


Extracellular protease VCPl


bsia50

bssugx

bspbya

btisra

btfini

btkurs

bcespr

biprot

ndapII

srespd

tscald

vmapk

ccalp2

abexpr

msthmy

opexpr

vcexp

1

saalpr

1-54

1-16

1-2 1

1-14 223-243

1-15

6-20

1-15 223-243

1-15

1-26

1-23

1-15

1-36

1-29

1-19

1-28 217-222

1-18 170-193

1-20

1-13

Strongin et al., 1978

Durham, 1993

Shimogaki et al., 1991

Chestukhina et al., 1986


Kunitate et al., 1989


Balaban et al. , 1994

Tsujibo et al., 1990

C Lavrenova et al., 1984

C Freeman et al., 1993

A Kwon et al., 1994

C Murphy

&

Walton, 1996

C Burton et al., 1993

C Gaucher

&

Stevenson, 1976

C Abraham

&

Breuil, 1995

C Segers et al., 1995

C Larcher etl., 1996

Table

4. Highest conserved residues in subtilases v = variability)

Residue

u = I u =

2

u = 3

Context/function Exception

32

34

64

65

68

69

70

83

90

125

152

154

155

189

193

20

1

219

220

22

1

223

225

229

G

S

G

N

Catalytic triad residue

Bend;

4,

@

=

99 , 179


Buried helix, close packing

Buried helix,

close

packing, directly under catalytic triad



Helix/turn, close packing

Buried fi-strand, hydrophobic packing to helix C

Bend, directly adjacent to catalytic triad

Lines

S 1

pocket

Lines

S I

pocket; 6,

=

114 , 163

Oxyanion stabilization

Turn

at surface, side chain turned into pocket

Begin turn

Bend at end &strand, hydrophobic ring stacks with H226

Bend between e9 and hF;

4, @ =

147 , 160

OD1 H -bonded to backbone NH -154




Buried helix,

close

packing

N (Islasp), A (smserp), P (smsspl, smssp2)

del (asaspa)

M (Islasp), I (sepepp, ddtagc)

G

(nahlys), T (bsb pf),

I

(sepepp)

T

(smstab), A (smsspl, paaf70)

A (Islasp),

T

(efcyla)

W (bsbpf), M (seepip)

P (Islasp), C (hakx2), T (acfurl, bcpc2)

M (sepepp), del (bssepr)

D (Ispc2, bcpc2, cepc2, hspc2)

del (sepepp), S

(smserp),

L (bspara)

Y (=pe w), D (dmpga9). T (vmvapt)

I (seepip, smstab)

N (sepepp, Ilnisp)

G (Islasp), S (sepepp, ddtagc)

T

(bssepr)


17/23

Subtilases

517

Large insertions and deletions

peptidases, and include large

N-

and C-terminal deletions. All but

one of the internal deletions can be readily accommodated by

The 190 residues that constitute the scrs, as defined from the connecting residues that are spatially adjacent in the 3D structures

known crystal structures (Siezen et al., 1991) and shown in Fig- of subtilisin/thermitase. Particularly interesting in this respect is

ure

2

are present in nearly all the subtilases. Som e unusual dele- the natural deletion of the Ca l-i on binding loop, residues 74-82,

tions are found, however, as listed in Table

5,

and this implies that in the

Enterococcus

subtilase (efcyla), thereby presumably extend-

not all of these core residues are essential for proper folding. Most ing helix

C

by another four residues (Booth et al., 1996); this is

of these deletions occur in subtilase family

D,

the lantibiotic leader precisely the loop deletion that was engineered into subtilisin to

Table 5. Large or unusual deletions and insertions

Unusual deletion

Missing residues Context Family Enzyme

1-13

65-66

14-82

96-102

180-189

257-215

N-terminus, hA

Part hC, adjacent catalytic His

Ca-binding loop + hC extended

Turn, substrate-binding region

Turns

C-terminus, hH

sepepp

asaspa

efcyla

smserp

sePePP

lslasp

Large insertion

Inserted residues

Position Number Properties Family Enzyme

vr5

vr6

v r l

vr8

V I 9

v r l l

vr13

vr15

vr16

vr18

vr19

N-term. Up to 98

59

34

vr

1

1 8

vr4 30-33

28-30

26-3 1

23

147-213

30

42

16

51

34

18

22

16-18

134-169

13-15

21

20-22

149

2 1

22

20

19

38

34

25

22-24

21

No homology

Highly charged

Highly charged

Weak homology

High homology

Medium homology, conserved S-S bond

?

High homology

Weak homology, see alignment in Fig.

5

Highly charged (50%)

Highly charged

High homology

Weak homology

Weak homology in central section (Fig.

5 )

High homology

Weak homology

S - S

bond ?

High homology

S-S bond ?

High homology

S-S bond?

E

F

C

C

F

B

F

F

F

F

F

C

F

F

F

F

D

F

F

F

D

A

E

F

F

B

E

E

B

F

F

Most family members

spscpa

scyct5

scyct5

spscpa, Ilprtp, ldprtb

dnbpr, dnavp2, dnavp5, xcproa,

alaprl

llspO9, atserp, cmc ucu, agserp,

lep69, paafl0

smssp l, smssp2

pfpyro, tsplst, dmpga9, hstpp2,

CetPP

PfPYro

siezen, 1997, subtilase

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