Thomas W. Jeffries

USDA, Forest Service, Forest Products Laboratory, Madison, WI 53705

Summary:

Kappa reduction and color removal are two consequences of treating kraft pulps with xylanases. They depend on the origin and processing of the pulp, access of enzymes to the xylan substrate, and the natures of the enzymes themselves. Kappa reduction has been attributed to removal of lignin. More recent research has implicated removal of hexeneuronic acid (HexA)-xylosaccharides as the basic mechanism for kappa reduction. HexA is formed from the 4-O-methyl-D-glucuronic acid component of xylan during kraft pulping. Research has shown that it accounts for a significant fraction of the oxidizable components removed by xylanase. In addition to hexeneuronic acid, xylanases release materials that absorb in the visible region (chromophores). Previous studies have suggested that these chromophoric materials are derived from lignin, but proton and carbon NMR have not indicated the presence of aromatic moieties in extracts. We have found that different xylanases release different amounts of HexA and chromophoric products from kraft pulps. Optimal xylanase treatment occurs when HexA and chromophoric moieties are released while yield losses from xylan removal are minimized. Alkaline active and extreme thermophilic enzymes are of particular interest for pulp treatment. Such xylanases are being commercialized for bleaching pulps and other applications. Although useful enzymes are now commercial, higher specific activity, and higher pH and temperature optima are desirable. New xylanases can be cloned from microbial or environmental samples by using direct PCR amplification based on conserved motifs. Primers appropriate for cloning various groups of xylanases can be identified through BLAST/PileUp analyses of known sequences. Moreover, motifs of enzymes with certain pH optima, substrate specificity or thermal stability can be identified then mutated or shuffled to create novel activities with desired traits. This paper describes a technique for identifying modular units or protein motifs useful for primer development and module shuffling.

Hemicelluloses are widely distributed heteropolysaccharides. The enzymes that degrade them are ubiquitous and diverse. In nature, xylans have L-arabinose, acetyl, glucuronic, 4-O-methylglucuronic, and p-coumaric side chains, and ferulic acid cross linkages. Intra-chain hydrogen bonding occurs through the O-3 position giving unsubstituted xylan a helical twist. Acetylation and substitution, however, disrupt and complicate that structure. Xylans are complexed with cellulose and pectin and are bound to lignin. As esterification and substitution increases, digestibility of hemicellulose decreases. Removal of the side chains is carried out by acetyl esterase ferulic esterase, glucuronosidase, or arabinosidase. Native xylans present a formidable substrate for degradation, but commercial xylans recovered by alkaline extraction or those that are present in kraft pulps have a low degree of substitution.

Viikari et al. first showed that fungal xylanases decrease the amount of bleach chemical required to attain a given brightness. Subsequent numerous advancements have moved the technology toward commercialization (see () and references therein). More recently, it has been shown that enzyme treatment reduces the pulp kappa number. The mechanism behind xylanase-enhanced bleaching is still being elucidated. One hypothesis is removal of xylan allows lignin extraction. However, xylanase activity, as measured by reducing sugar release, does not correlate well with pulp brightness or the decreased chemical demand brought about by a given enzyme treatment. Previous work by our group demonstrated the liberation from pulps of materials having UV absorption at ca. 237 and 260 nm, and that liberation of material absorbing at 237 correlates well with enhanced brightness following bleaching. Different purified xylanase isozyme fractions release UV-absorbing materials to different extents, and in some instances, activities of the isozymes are synergistic.

Desirable characteristics of xylanases for bleaching include the following:

Microbial xylanases fall into two main families of glycosyl hydrolases. These are family F (previously known as 10) and G (previously known as 11). Both use ion pair catalytic mechanisms and both retain anomeric configuration following hydrolysis. Family 10 xylanases are larger, more complex and more diverse; family 11 are smaller, more consistent in structure and more specific for xylan. We have therefore focused on identifying novel enzymes with high specificity for the HexA and chromophoric side chains. The first step in this process is to determine which or other constituent motifs of cloned enzymes determine substrate specificity, pH optimum or kinetic properties.

Protein sequences for family 10 and family 11 xylanases are drawn from the SWISS-PROT database. One may refer to specific listings such as Amos Bairoch's site listing glycosyl hydrolase families (Table 1), or alternatively, once one has identified a cloned enzyme with appropriate properties (such as pH optimum, HexA specificity, chromophore release) it is possible to perform a BLAST/PileUp analysis to identify how it is similar to (or differs from) other enzymes in this same group. Starting from a "Query" protein sequence of an enzyme with appropriate thermal, pH or kinetic characteristics, one performs a basic BLAST search. This returns a set of "Subject" segments of various proteins with high similarity or identity to regions of the query protein (Fig. 1).

A B C

Figure 1. Overall process for carrying out a BLAST/PileUp segment analysis

One then designates each of the subject segments with an appropriate notation related to its origin and alignment and enters each as a separate peptide file into the database. A program such as PileUp from the GCG Sequence Analysis Software Package can then be used to align each of the peptide segments against the initial query protein. An example of such an alignment for a BLAST/PileUp analysis of Bacillus pumilus XynA (XYNA_BACPU) is shown in Figure 2. Note that this search returned peptide segments having similarity to 27 family 11 xylanases, only a small portion of the total report is shown here.

sp|P55335|XYN2_MAGGR ENDO-1,4-BETA-XYLANASE 22 PRECURSOR (XYLANASE 22)

(1,4-BETA-D-XYLAN XYLANOHYDROLASE 22)

Length = 233

Score = 232 (106.8 bits), Expect = 1.5e-65, Sum P(5) = 1.5e-65

Identities = 42/83 (50%), Positives = 57/83 (68%)

Query:   137 KGSFYADGGTYDIYETTRVNQPSIIGIATFKQYWSVRQTKRTSGTVSVSAHFRKWESLGM 196
             +G+  A GGTY ++E+TRVNQPSI G  TF+QYW++RQ KR SGTV+    F+ WE  GM
Sbjct:   145 RGTLQAAGGTYTLHESTRVNQPSIEGTRTFQQYWAIRQQKRNSGTVNTGEFFQAWERAGM 204

Query:   197 PMGKMYETAFTVEGYQSSGSANV 219
              MG         EGY+S+G++N+
Sbjct:   205 RMGNHNYMIVATEGYRSAGNSNI 227

 Score = 130 (59.9 bits), Expect = 1.5e-65, Sum P(5) = 1.5e-65
 Identities = 19/29 (65%), Positives = 26/29 (89%)

Query:   104 GNSYLCVYGWTQSPLAEYYIVDSWGTYRP 132
             GN+YLC+YGWTQ+PL EYYI++++G Y P
Sbjct:   110 GNAYLCIYGWTQNPLVEYYILENYGEYNP 138

 Score = 54 (24.9 bits), Expect = 1.5e-65, Sum P(5) = 1.5e-65
 Identities = 11/33 (33%), Positives = 14/33 (42%)

Query:    28 RTITNNEMGNHSGYDYELWKDYGNTSMTLNNGG 60
             R  T +  G H+GY Y  W D  +     N  G
Sbjct:    39 RQSTPSSTGRHNGYYYSWWTDGASPVQYQNGNG 71

 Score = 49 (22.6 bits), Expect = 1.5e-65, Sum P(5) = 1.5e-65
 Identities = 9/20 (45%), Positives = 13/20 (65%)

Query:    58 NGGAFSAGWNNIGNALFRKG 77
             NGG++S  W + GN +  KG
Sbjct:    70 NGGSYSVQWQSGGNFVGGKG 89

 Score = 43 (19.8 bits), Expect = 1.5e-65, Sum P(5) = 1.5e-65
 Identities = 8/16 (50%), Positives = 11/16 (68%)

Query:    90 GNISINYNASFNPGGN 105
             G+ SI Y+ +FNP  N
Sbjct:    94 GSKSITYSGTFNPVNN 109

 Score = 33 (15.2 bits), Expect = 7.7e-50, Sum P(3) = 7.7e-50
 Identities = 6/14 (42%), Positives = 8/14 (57%)

Query:    49 YGNTSMTLNNGGAF 62
             Y  T   +NNG A+
Sbjct:   100 YSGTFNPVNNGNAY 113

Figure 2. Subject segments of the fifth-most closely related enzyme (XYN2_MAGGR) returned by a BLAST analysis of the XYNA_BACPU protein.

Peptide segments with scores greater than 50 will generally line up properly in the PileUp analysis. Segments with scores of 35 or less generally will not align properly. Note that the subject segment generally occurs in about the same position as the query segment, but in some instances, it may be later or earlier in the protein sequence. This is particularly true of proteins such as xylanases which have multiple domains that can be inverted or duplicated.

Although inspection of a single set of subject segments will provide some idea about sequence similarities, the compilation of subject segments from multiple enzymes is far more informative. Because the BLAST analysis returns segments in the order that they are most closely related to the Query protein, it is possible to start with any one enzyme in the family as a query sequence and align the most closely related subject segments to it. This will identify the conserved sequences in that taxonomic group or cluster. Oligonucleotide probes based on the most conserved amino acid sequences in the motifs can then be designed for direct PCR. The derived PCR fragments are useful as probes for cloning from libraries or as starting points for cycle sequencing.

In the analysis shown (Fig. 3), the Xyna_Bacpu protein sequence runs the entire length of the alignment (228 amino acids). The family 11 enzymes from Streptomyces lividans (Xynb_Stri and Xync_Stri) are most distantly related to Xyna_Bacpu in this analysis, and hence the segments are farther away than the segments from others (e.g., Xyna_Cloab). In some regions of the protein, (e.g. positions 1-30) only one or two other proteins have any sequence similarity, and even then the similarity is slight, whereas in other regions, such as the domain encompassing the active site nucleophile at position 120, all of the analyzed enzymes are represented by subject segments.

Segment PileUp analysis has several advantages over a normal PileUp of complete proteins: First, the alignments are much more efficient. Because the BLAST analysis returns only segments having high sequence similarity, no gaps are introduced. This makes the alignment run much more rapidly. Even though these analyses can employ 70 or 100 segments in a single analysis, the computation time is insignificant in most instances.

Second, the conserved motifs can be readily visualized. For example, simple inspection of the region from position 90 to 130 shows that this is a block of the protein that is conserved from bacteria to fungi. It is not necessary to pick out or block out the unrelated segments. Likewise inspection of Fig. 3 from position 135 to 181 shows that this is a region that has about five major variations and a conserved block from 171-181. These groupings are reflected in the PileUp cluster diagram as the 27 related segments in the center of the figure.

Third, and most importantly, a global alignment need not be attained with the whole protein sequence. Individual Subject segments are free to align with other the Query protein and other Subject segments according to the relative similarities within the motif. One Subject segment (depicting one motif) can be more closely related to the Query protein, and another Subject segment (depicting a second motif) can be less related, and they will each align independently. Their relative proximities to the Query protein displays their relative relatedness to the two motifs. This can be most readily observed by comparing the relatedness of the aligned segments surrounding the conserved active site nucleophile at position 121 to the relatedness of the segments at the active site proton donor at position 210. Note that the Xyna_Rumfl_86 segment is closest to the Xyna_Bacpu protein at the nucleophile site but the Xyna_Rumfl_213 segment which corresponds to the proton donor domain from the same Xyna_Rumfl protein, is further away. This may reflect the relative electronegativity of the ion pair in these two instances.

The clustering of the motifs can also be observed by converting the .msf file into a graphic as shown in Fig. 4. Note, however, that the clusters depicted are representative of structural features rather than taxonomic relatedness.

Using different enzymes as the Query protein can return somewhat different results. For example, proteins related to the Xync_Strli enzyme are known to release relatively large amounts of HexA and chromophores. However, the first 63 amino acids of this 240 amino acid protein bear no significant similarity to any of the other family 11 proteins in SWISS_PROT (Fig. 5). Between positions 64 and 85, however, there is a highly conserved block with two motifs reflecting sequences found in bacterial and fungal proteins. If one compares this region to the position 30-78 region of Xyna_Bacpu, it would appear that in the Xyna_Bacpu protein, the motif has two segments: one that is more closely related to the bacterial form (30 to 50), and then one more closely related to the fungal enzymes (from 51-75). Other differences can also be seen in the regions surrounding the nucleophile and the proton donor. Using very distantly related family 11 enzymes such as that from Fibrobacter succinogenes (XYNC_FIBSU) as the Query protein yields a very different result. A great portion of this large enzyme has no similarity to other family 11 proteins, and a nucleophile is found around position 409. One of the proton donor sites is at position 501, but its surrounding motif possesses so little similarity to most family 11 enzymes that it was not readily identified in this analysis.

Figure 5. PileUp of: Xync_Strli

                1                                                   50
    Xync_Strli  MQQDGTQQDR IKQSPAPLNG MSRRGFLGGA GTLALATASG LLLPGTAHAA 
                51                                                 100
 Xyn1_Emeni_83  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~GSYSTQWT NCGNFVAGKG 
    Xync_Strli  TTITTNQTGT DGMYYSFWTD GGGSVSMTLN GGGSYSTQWT NCGNFVAGKG 
 Xynb_Strli_64  ~~~~~~~~~~ ~~~YYSFWTD SQGTVSMNMG SGGQYSTSWR NTGNFVAGKG 
 Xynd_Celfi_64  ~~~~~~~~~~ ~~~FYSFWTD SPGSVSMDLN SGGGY.TRWS NTGNFVAGKG 
 Xyna_Bacci_64  ~~~~~~~~~~ ~~~YWQNWTD GGGIVNAVNG SGGNYSVNWS NTGNFVVGKG 
 Xyna_Bacsu_64  ~~~~~~~~~~ ~~~YWQNWTD GGGIVNAVNG SGGNYSVNWS NTGNFVVGKG 
Xyna_Bacci_139  ~~~~~~~~~~ ~~DYWQNWTD GGGIVNAVNG SGGNYSV~~~ ~~~~~~~~~~ 
Xyna_Bacsu_139  ~~~~~~~~~~ ~~DYWQNWTD GGGIVNAVNG SGGNYSV~~~ ~~~~~~~~~~ 
 Xyna_Bacst_64  ~~~~~~~~~~ ~~~YWQYWTD GGGMVNAVNG PGGNYSVTWQ NTGNFVVGKG 
 Xyn2_Aspng_64  ~~~~~~~~~~ ~~~YYSFWTD GGGDVTYTNG DAGAYTVEWS NVGNFVGGKG 
  Xynb_Aspa_64  ~~~~~~~~~~ ~~~YYSFWTD GGGDVTYTNG NAGSYSVEWS NVGNFVGGKG 
 Xyn1_Emeni_64  ~~~~~~~~~~ ~~~YYSFWTD GGGDVTYTNG AGGSYTVQWS NVGNFVGGKG 
 Xyn2_Emeni_64  ~~~~~~~~~~ ~~~YYSFWTD GGGDVTYTNG DGGSYTVEWT KVGNFVGGKG 
 Xyn2_Aspng_76  ~~~~SSTGEN NGFYYSFWTD GGGDVTYTNG DAGAYTVEWS NVGNFVG~~~ 
  Xynb_Aspa_76  ~~~~SSTGEN NGYYYSFWTD GGGDVTYTNG NAGSYSVEWS NVGNFVG~~~ 
 Xyn2_Emeni_76  ~~~~SSTGTS GGYYYSFWTD GGGDVTYTNG DGGSYTVEWT KVGNFVG~~~ 
 Xyn1_Cocca_64  ~~~~~~~~~~ ~~~FWSWWSD GGARATYTNG AGGSYSVSWG SGGNLVGGKG 
 Xyn2_Maggr_64  ~~~~~~~~~~ ~~~YYSWWTD GASPVQYQNG NGGSYSVQWQ SGGNFVGGKG 
 Xyn1_Humin_64  ~~~~~~~~~~ ~~~FYSWWSD GGGQVQYTNL EGSRYQVRWR NTGNFVGGKG 
 Xyna_Bacpu_73  ~~~~~~~~~~ ~~~~~~~~~~ ~~GNTSMTLN NGGAFSAGWN NIGNALFRKG 
                101                                  *             150
 Xyn1_Emeni_83  WSTGD.GNVR YNGYFNPVG~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
    Xync_Strli  WSTGD.GNVR YNGYFNPVGN GYGCLYGWTS NPLVEYYIVD NWGSYRPTGT 
 Xynb_Strli_64  WANG~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Xynd_Celfi_64  WSTGGRKTVS YSGQFNPSRN AYLTLYGWTQ SPLVEYYIVD SWGTYRPTGT 
 Xyna_Bacci_64  WTTG~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Xyna_Bacsu_64  WTTG~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Xyna_Bacst_64  WTVGSPN~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Xyn2_Aspng_64  WNPG~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
  Xynb_Aspa_64  WNPG~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Xyn1_Emeni_64  WNPG~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Xyn2_Emeni_64  WNPG~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Xyn1_Cocca_64  WNPG~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Xyn2_Maggr_64  WMPG~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Xyn1_Humin_64  WNPGTG~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
Xynb_Strli_104  ~~~~GRRTVQ YSGSFNPSGN AYLALYGWTS NPLVEYYIVD NWGTYRPTGE 
Xyn2_Aspng_148  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~GT 
 Xynb_Aspa_148  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~GT 
Xyn1_Emeni_148  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~GQ 
Xyn2_Emeni_148  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~GT 
Xyna_Bacci_112  ~~~~~~~~~~ ~~GVWAPNGN GYLTLYGWTR SPLIEYYVVD SWGTYRPTGT 
Xyna_Bacsu_112  ~~~~~~~~~~ ~~GVWAPNGN GYLTLYGWTR SPLIEYYVVD SWGTYRPTGT 
Xyna_Bacst_112  ~~~~~~~~~~ ~~GIWEPSGN GYLTLYGWTR NALIEYYVVD SWGTYRPTGN 
Xyn2_Aspng_107  ~~~~~~~DIT YSGTFTPSGN GYLSVYGWTT DPLIEYYIVE SYGDYNP~~~ 
 Xynb_Aspa_107  ~~~~~~~DIT YSGNFTPSGN GYLSVYGWTT DPLIEYYIVE SYGDYNP~~~ 
Xyn2_Emeni_108  ~~~~~~~~IS YSGSFIPSGN GYLSVYGWTQ NPLIEYYIVE SYGDYNP~~~ 
Xyn1_Emeni_108  ~~~~~~~~IN YGGSFNPSGN GYLAVYGWTQ NPLIEYYIVE SYGTYNP~~~ 
Xyn1_Humin_108  ~~~~~~~~IN YGGYFNPQGN GYLAVYGWTR NPLVEYYVIE SYGTYNP~~~ 
Xyn2_Maggr_118  ~~~~~~~~~~ ~~~~~~~~GN AYLCIYGWTQ NPLVEYYILE NYGEYNP~~~ 
Xyn1_Cocca_108  ~~~~~~~~IT YSGTYNYNGN SYLAVYGWTR NPLVEYYVVE NFGTYDPS~~ 
Xyna_Bacpu_104  ~~~~GNISIN YNASFNPGGN SYLCVYGWTQ SPLAEYYIVD SWGTYRPTGA 
Xyn2_Maggr_104  ~~~~GSKSIT YSGTFNPVNN G~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
                151                                                200
Xyn1_Cocca_195  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~SGSVN 
Xyn1_Humin_196  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~GSVN 
Xyna_Bacpu_195  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~SGTVS 
Xyn2_Maggr_195  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~SGTVN 
Xyn2_Aspng_196  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~GTVT 
 Xynb_Aspa_196  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~GTVT 
Xyn1_Emeni_196  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~GTVT 
Xyn2_Emeni_195  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~SGSVT 
Xynd_Celfi_196  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~GTIT 
Xyn2_Maggr_220  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
Xynb_Strli_196  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~GTIT 
Xyna_Bacci_187  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~QSK RPTGSNATIT 
Xyna_Bacsu_187  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~QSK RPTGSNATIT 
Xyna_Bacst_187  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~QSK RPTGSNVSIT 
    Xync_Strli  YKGTVSSDGG TYDIYQTTRY NAPSVEGTKT FQQYWSVRQS KVTSGSGTIT 
 Xynd_Celfi_64  FMGTVTSDGG TYDIYRTQRV NKPSIEG~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
Xyna_Bacci_179  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~T FTQYWSVRQS KRPTGSNATI 
Xyna_Bacsu_179  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~T FTQYWSVRQS KRPTGSNATI 
Xynb_Strli_104  YKGTVTSDGG TYDIYKTTRV NKPSVEGTRT FDQYWSVRQS KRTGGTIT~~ 
Xynd_Celfi_177  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~SST FYQYWSVRQQ KRTGGTIT~~ 
Xyn2_Aspng_148  YKGTVTSDGS VYDIYTATRT NAASIQGTAT FTQYWSVRQN KRVGGTVT~~ 
 Xynb_Aspa_148  TRGNVSSDGS VYDIYTATRT NAPSIQGTAT FSQYWSVRQN KRVGGTVT~~ 
Xyn1_Emeni_148  HRGTVYSDGA TYDIYTATRY NAPSIEGTAT FEQFWSVRQS KRTGGTVT~~ 
Xyn2_Emeni_148  HQGTLESDGS TYDIYTATRE NAPSIEGTAT FTQFWSVRQS KRTSGSVT~~ 
Xyn1_Cocca_151  ~KGTVTSDGS SYKIAQSTRT NQPSIDGTRT FQQYWSVRQN KRSSGSVNMK 
Xyn1_Humin_150  YKGTFYTDGD QYDIFVSTRY NQPSIDGTRT FQQYWSIRKN KRVGGS~~~~ 
Xyn2_Maggr_151  ~RGTLQAAGG TYTLHESTRV NQPSIEGTRT FQQYWAIRQQ KRNSGT~~~~ 
Xyna_Bacci_112  YKGTVKSDGG TYDIYTTTRY NAPSIDGDRT ~~~~~~~~~~ ~~~~~~~~~~ 
Xyna_Bacsu_112  YKGTVKSDGG TYDIYTTTRY NAPSIDGDRT ~~~~~~~~~~ ~~~~~~~~~~ 
Xyna_Bacst_112  YKGTVNSDGG TYDIYTTMRY NAPSIDGTQT FQQFWSVRQS KRPTGSNVSI 
Xyna_Bacpu_104  YKGSFYADGG TYDIYETTRV NQPSIIGIAT FKQYWSVRQT KRTSGTVSVS 
                201                         •             241
Xyn1_Cocca_195  MKTHFDAWAS KGMNLGQHYY QIV~~~~~~~ ~~~~~~~~~~ ~
Xyn1_Humin_196  MQNHFNAWQQ HGMPLGQHYY QVV~~~~~~~ ~~~~~~~~~~ ~
Xyna_Bacpu_195  VSAHFRKWES LGMPMGK~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~
Xyn2_Maggr_195  TGEFFQAWER AGMRMGNHNY MIV~~~~~~~ ~~~~~~~~~~ ~
Xyn2_Aspng_196  TSNHFNAWAK LGMNLGTH.N YQIVATEGYQ SSGSSS~~~~ ~
 Xynb_Aspa_196  TSNHFNAWAK LGMNLGTH.N YQILATEGYQ SSGSSS~~~~ ~
Xyn1_Emeni_196  TANHFNAWAA LGMRLGTH.N YQIVATEGYQ SSGSAS~~~~ ~
Xyn2_Emeni_195  TQNHFDAWSQ LGMTLGTH.N YQIVAVEGYQ SSGSAS~~~~ ~
Xynd_Celfi_196  SGNHFDAWAS KGMNLGRH.N YMIMATEGYQ SSGSSS~~~~ ~
Xyn2_Maggr_220  ~~~~~~~~~~ ~~~~~~~~~~ YMIVATEGYR SAGNSNINV~ ~
Xynb_Strli_196  TGNHFDAWAR AGMPLGNFSY YMIMATEGYQ SSGTSSINVG G
Xyna_Bacci_187  FTNHVNAWKS HGMNLGSNWA YQVMATEGYQ SSGSSNVTV~ ~
Xyna_Bacsu_187  FSNHVNAWKS HGMNLGSNWA YQVMATEGYQ SSGSSNVTV~ ~
Xyna_Bacst_187  FSNHVNAWRS KGMNLGSSWA YQVLATEGYQ SSGRSNVTV~ ~
Xyn1_Emeni_220  ~~~~~~~~~~ ~~~~~~~~~~ YQIVATEGYQ SSGSASITV~ ~
Xyn2_Aspng_220  ~~~~~~~~~~ ~~~~~~~~~~ YQIVATEGYQ SSGSSSITV~ ~
 Xynb_Aspa_220  ~~~~~~~~~~ ~~~~~~~~~~ YQILATEGYQ SSGSSSITI~ ~
Xyn2_Emeni_220  ~~~~~~~~~~ ~~~~~~~~~~ YQIVAVEGYQ SSGSASITVS ~
Xynd_Celfi_220  ~~~~~~~~~~ ~~~~~~~~~~ YMIMATEGYQ SSGSSSITVS ~
Xyn1_Humin_219  ~~~~~~~~~~ ~~~~~~~~~Y YQVVATEGYQ SSGESDIYV~ ~
Xyn1_Cocca_219  ~~~~~~~~~~ ~~~~~~~~~Y YQIVATEGYF STGNAQITVN ~
Xyna_Bacpu_219  ~~~~~~~~~~ ~~~~~~~~~Y ETAFTVEGYQ SSGSANV~~~ ~
    Xync_Strli  TGNHFDAWAR AGMNMGQFRY YMIMATEGYQ SSGSSNITVS G

Analysis of a family 10 xylanase (xylanase A from Streptomyces lividans) shows several "motifs" outside those surrounding the proton donor (at position 169) and the nucleophile (at position 277).

Figure 6. PileUp of: Xlna_Strli (Stmxlna)

             1                                                   50
    Stmxlna  MGSYALPRSG VRRSIRVLLA ALVVGVLGTA TALIAPPGAH AAESTLGAAA 
  U41627_33  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~A AGVVALAGTA QAAGALGDAA 
  L11080_42  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~AATTLKEAA 
  U08894_16  ~~~~~~~~~~ ~~~~~~~~LL AGAVGLLTAA AALVAPSPAV AAESTLGAAA 
  U41627_20  ~~~~~~~~~~ ~~~~~~RCAL SLLTAGVLAA AGVVALAGTA QAAGALGDAA 
             51                                                 100
    Stmxlna  AQSGRYFGTA IASGRLSDST YTSIAGREFN MVTAENEMKI DATEPQRGQF 
  U41627_33  AAKGRYFGAA VAANHLGEAA YASTLDAQFG SVTPENEMKW DAVESSRNSF 
 Z69782_370  ~~~~~~~GVA VPYRALTNPV DVAFIKRHFN SITAENEMKP EALEPIEDNF 
 Z69782_381  ~~~~~~~~~~ ~~~~~~~~PV DVAFIKRHFN S~~~~~~~~~ ~~~~~~~~~~ 
 Z46264_381  ~~~~~~~GVA LPSKVFLNPK DIELITKHFN SITAENEMK~ ~~~~~~~~~~ 
 Z46945_377  ~~~~~~~GVA LPSKVFINQK DIALISKHSN SSTAENEMKP DS~~~~~~~~ 
 Z50866_263  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~VWGPRG 
  L11080_42  DGAGRDFGFA LDPNRLSEAQ YKAIADSEFN LVVAENAMKW DATEPSQNSF 
     2exo_2  DGAGRDFGFA LDPNRLSEAQ YKAIADSEFN LVVAENAMKW DATEPSQNSF 
  Z49894_40  ~~~~~YFGTC SDQALLQNSQ NEAIVASQFG VITPENSMKW DALEPSQGNF 
  S57397_40  ~~~~~YFGTC SDQALLQNSQ NEAIVASQFG VITPENSMKW DALEPSQGNF 
  P33559_55  ~~~~~~~~~~ ~~~~~~~NSK TPAVIKADFG ALTPENSMKW DATEPSRGQF 
 Z46264_426  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~KF 
 Z46945_422  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~KF 
 U27183_382  ~~~~~~~~~~ ~~~~~~~~~~ HAQLTARHFN MLAAANAMKP ESLQPTEGNF 
 Z50866_434  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~KHYD QITAENAMKP ESIQPTEGAF 
  U08894_16  AQSGRYFGTA IASGRLNDST YTTIANREFN MVTAENEMKI DATEPNRGQF 
  U41627_20  AAK~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
             101                                                150
    Stmxlna  NFSSADRVYN WAVQNGKQVR GHTLAWHSQQ PGWMQSLSGR PLRQAMIDHI 
 U27183_461  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~LRQRLSTHI 
 Z50866_510  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~LRDRMRTHI 
 Z69782_465  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~LLERLKKYI 
 Z46264_470  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~LSKE AMTERLKEYI 
 Z46945_466  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~LSKE EMTERLREYI 
 P33559_130  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~LIEVMKNHI 
 Z49894_127  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~LRSVMTNHI 
 S57397_127  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~LRSVMTNHI 
  U41627_33  SFSAADRIVS HAQSKGMKVR GHTLVWHSQL PGWVSPLAAT DLRSAMNNHI 
 Z69782_370  NFSIADEYLN FCRKNNIAIR GHNL~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z50866_263  DATVTLSDDA HGGAQAALVA GRTQAWHGIG ATVTDVFQTG RTYT~~~~~~ 
 Z50866_287  ~~~~~~~~~~ ~~~~~~~~VA GRTQAWH~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
  L11080_42  SFGAGDRVAS YAADTGKELY GHTLVWHSQL PDWAKNLNGS AFESAMVNHV 
     2exo_2  SFGAGDRVAS YAADTGKELY GHTLVWHSQL PDWAKNLNGS AFESAMVNHV 
 U41627_178  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
  Z49894_40  GWSGADYLVD YATQHNKKVR GHTLVWHSQL PSWVSSI~~~ ~~~~~~~~~~ 
  S57397_40  GWSGADYLVD YATQHNKKVR GHTLVWHSQL PSWVSSI~~~ ~~~~~~~~~~ 
  P33559_55  SFSGSDYLVN FAQSNNKLIR GHTLVWHSQL PSWVQAITDK ~~~~~~~~~~ 
 Z46264_426  RFETADKYIQ FVEENGMVIR GHTLVWHNQT PDW~~~~~~~ ~~~~~~~~~~ 
 Z46945_422  RFETADKYIE FAQQNGMVVR GHTLVWHNQT PEW~~~~~~~ ~~~~~~~~~~ 
 U27183_382  AFDNADKIVD YAIAHNMKMR GHTLLWHNQV PDWV~~~~~~ ~~~~~~~~~~ 
 Z50866_434  TFEAGDALID SAVRNGQRVY GHTLVWHSQT PDW~~~~~~~ ~~~~~~~~~~ 
  U08894_16  NFSSADRIYN WAVQNGKQVR GHTLAWHSQQ PGWMQSLSGS SLRQAMIDHI 
             151                •                               200
    Stmxlna  NGVMAHYKGK IVQWDVVNEA FADGSSGARR DSNLQRSGND WIEVAFRTAR 
 U27183_461  TAVLEHIQTK CGSLDPI~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z50866_510  ETVADHYRQK ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z50866_539  ~~~~~~~~~~ IVAYDVVNEA IAESESDGLR RS~~~~~~~~ ~~~~~~~~~~ 
 Z69782_465  QTVVSRYKGR IYAWDVVNEA IDENEPDGFR RSD~~~~~~~ ~~~~~~~~~~ 
 Z46264_470  HTVVGHFKGK VYAWDVVNEA ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z46945_466  HTVVGHFKGK VYAWDVVNEA ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 U27183_489  ~~~~~~~~~~ ~~~WDVVNEV LDESGN~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 P33559_130  TTVMQHYKGK IYAWDVVNEI FNEDGS~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z49894_127  NEVVGRYKGK IMHWDVVNEI F~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 S57397_127  NEVVGRYKGK IMHWDVVNEI F~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
  U41627_33  TQVMTHYKGK IHSWDVVNEA FQDGGSGARR SSPFQ~~~~~ ~~~~~~~~~~ 
  L11080_42  TKVADHFEGK VASWDVVNEA FADGGGRRQD SAFQQKLGNG YIETAFRAAR 
     2exo_2  TKVADHFEGK VASWDVVNEA FADGGGRRQD SAFQQKLGNG YIETAFRAAR 
 U41627_178  ~~~~~~~~~~ ~~~~~~~~~~ ~~~GSGARRS SPFQDKLGNG FIEEAFRTAR 
 Z49894_172  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~GED FVRIAFETAR 
 S57397_172  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~GED FVRIAFETAR 
 P33559_175  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~GDD YVRIAFETAR 
 Z46264_511  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~GLRR STWYQIMGPD YIELAFKFAR 
 Z46945_507  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~GLRR STWYQIMGPD YIELAFKFAR 
 Z69782_501  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~GFRR SDWFNVLGPE YIEKAFIYAH 
 Z50866_554  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~SDGLRR SRWFEVLGEQ YLDLAFQYAS 
 U27183_497  ~~~~~~~~~~ ~~~~~~~~~~ ~~DESGNLRN PTWLQIIGPD YIDKASEYAH 
  U08894_16  NGVMAHYKGK IVQWDVVNEA FADGNSGGRR DSNLQRTGND WIEVAFRTAR 
             201                                                250
    Stmxlna  AADPSAKLCY NDYNVENWTW AKTQAMYNMV RDFKQRGVPI DCVGFQSHFN 
  L11080_42  AADPTAKLCI NDYNVE~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
     2exo_2  AADPTAKLCI NDYNVE~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 U41627_178  TVDADAKLCY NDYNTD~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z49894_172  AADPDAKLYI NDYNLDSASY AKTQAMASYV KKWLAEGVPI DGIGSQAHYS 
 S57397_172  AADPDAKLYI NDYNLDSASY AKTQAMASYV KKWLAEGVPI DGIALSSLAN 
 P33559_175  AADPNAKLYI NDYNLDSASY PKLAGMVSHV KKWIEAGIPI DGIGSQTHLS 
 Z50866_588  ~~DGPVQLFL NDYNTE~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z46264_511  EADPDAKLFY NDYN~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z46945_507  EADPNAKLFY NDYN~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z69782_501  QADPNAQLFY NDYSTEN~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z50866_554  QA~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 U27183_497  EGDPSMTSFI TYHNIEN~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z46264_571  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~I DGIGMQCHIS 
 Z46945_567  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~I DGIGMQCHIS 
 U41627_224  ~~~~~~~~~~ ~~~~~~~~~~ AKSNAVYEMV KDFKQRGVPI DCVGFQSHFN 
 Z50866_604  ~~~~~~~~~~ ~~~~~~~~~~ AKRRAMLDVV NRLLARDVPV DGLGHQFHVN 
 Z50866_606  ~~~~~~~~~~ ~~~~~~~~~~ ~~RRAMLDVV NRLLA~~~~~ ~~~~~~~~~~ 
 L11080_220  ~~~~~~~~~~ ~~~~~~~~~~ AKSNSLYDLV KDFKARGVPL DCVGFQSHLI 
   2exo_178  ~~~~~~~~~~ ~~~~~~~~~~ AKSNSLYDLV KDFKARGVPL DCVGFQSHLI 
  U08894_16  NADPNAKLCY NDYNIENWNW AKTQGVYNMV RDFKQRGVPI DCVGFQSHFN 
 Z46264_554  ~~~~~~~~~~ ~~~~~~~~~~ ~KRDIIYNLV KDLKEKGL~~ ~~~~~~~~~~ 
 Z46945_550  ~~~~~~~~~~ ~~~~~~~~~~ ~KRDIIYNLV KSLKEKGL~~ ~~~~~~~~~~ 
 Z69782_544  ~~~~~~~~~~ ~~~~~~~~~~ ~KREYIYKLI KSLREKGIPI HGVGLQCHIS 
 U27183_544  ~~~~~~~~~~ ~~~~~~~~~~ ~KTQAMYDLV KKLKNEGVPI NGIGMQMHIS 
             251                         *                      300
 L11080_293  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~AADYKK VVQACMQVTR 
   2exo_251  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~AADYKK VVQACMQVTR 
 U41627_289  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~AQAANYTK VVNACLAVTR 
 Z49894_257  ~~~~~~~~~~ ~~~~~~~~~~ ~EVAITELDI AGAASSDYLN LLNACLNEQK 
 S57397_239  ~~~~~~~~~~ ~~~~~~~~~~ ~EVAITELDI AGAASSDYLN LLNACLNEQK 
 P33559_258  ~~~~~~~~~~ ~~~~~~~~~~ ~EIAVTELDI AGASSTDYVE VVEACLDQPK 
 Z50866_680  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~YAD VFDMLRQYPD 
    Stmxlna  SGSPYNSNFR TTLQNFAALG VDVAITELDI QGAPASTYAN VTNDCLAVSR 
 Z49894_172  SSHWSSTEAA GALSSLANTG V~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 S57397_172  TG~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 P33559_175  AG~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z46264_571  LATDIKQIEE AIKKFSTIPG IEIHITELDM ~~~~~~~~~~ ~~~~~~~~~~ 
 Z46945_567  LATDIRQIEE AIKKFSTIPG IEIHITELDI ~~~~~~~~~~ ~~~~~~~~~~ 
 Z69782_593  ~~~~~~~~~~ ~~~~~~~~~G IKIHVTEIDI SVA~~~~~~~ ~~~~~~~~~~ 
 L11080_256  ~~~~~~~DFR QNLQRFADLG VDVRITELDI R~~~~~~~~~ ~~~~~~~~~~ 
  2exo__214  ~~~~~~~DFR QNLQRFADLG VDVRITELDI R~~~~~~~~~ ~~~~~~~~~~ 
 U41627_224  SNSPVPSDFQ ANLQRFADLG VDVQITELDI EGSGSAQAAN YT~~~~~~~~ 
 U27183_579  ~~~~~~~NIK ASIEKLASLG VEIQVTELDM ~~~~~~~~~~ ~~~~~~~~~~ 
 Z50866_639  ~~~~~~SQMK ASIDAFATTG LLQAVTELD~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z50866_604  LLQP~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 L11080_220  VG~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
   2exo_178  VG~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
  U08894_16  SGSPYNSNFR TTLQNFAALG VDVAITELDI QGASPTTYAN VVNDCLAVSR 
 Z69782_544  VSWPSVEEVE KTIKLFSSI~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 U27183_544  INS~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
             301                                                350
 L11080_326  ~~~~~~~~~~ ~~DVFPGEGA ALVWDASYAK KPAYAAVMEA FGASPTPTP~ 
   2exo_284  ~~~~~~~~~~ ~~DVFPGEGA ALVWDASYAK KPAYAAVMEA ~~~~~~~~~~ 
 Z46264_668  ~~~~~~~~~~ ~~~~~RRNDW PLIFDKDHQA KLAYWAIV~~ ~~~~~~~~~~ 
 Z46945_644  ~~~~~~~~~~ ~~~~~RRNDW PLIFDKDYQA KLAYWAIV~~ ~~~~~~~~~~ 
 U27183_654  ~~~~~~~~~~ ~~~~~~~~~~ PLLFDSKLQA KPAYWAIVD~ ~~~~~~~~~~ 
 Z50866_709  ~~~~~~~~~~ ~~~~~~~EGA PLPFDDDLQA KPAFWGIVD~ ~~~~~~~~~~ 
 L11080_293  CQGVTVWGIT DKYSWVPDVF P~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
   2exo_251  CQGVTVWGIT DKYSWVPDVF P~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 U41627_289  CTGITVWGVT DKYSWRSGGT PLLFDGDYNK KPAYDAVLAA LGG~~~~~~~ 
 Z46264_653  ~~~VTFWGLK DDYSWRA~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z46945_649  ~~~VTFWGLK DDYSWRA~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 Z69782_646  ~~~VSFWGLK DDYSWLQGDF PLLFDKDYQP KFAFWSLID~ ~~~~~~~~~~ 
 Z49894_257  CVGITVWGVS DKDSWRASDS PLLFDGNYQP KDAYNAIVNA LS~~~~~~~~ 
 S57397_239  CVGITVWGVS DKDSWRASDS PLLFDGNYQP KDAYNAIVNA LS~~~~~~~~ 
 P33559_258  CIGITVWGVA DPDSWRSSST PLLFDSNYNP KPAYTAIANA L~~~~~~~~~ 
 Z50866_680  LFSVTLWGPY DSRSWR~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
 U08894_341  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~GGTSEPPPA 
 U08894_342  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~GTSEPPPA 
    Stmxlna  CLGITVWGVR DSDSWRSEQT PLLFNNDGSK KAAYTAVLDA LNGGDSSEPP 
 Z50866_574  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~AFQYASQA FNGGDTDGP~ 
   U08894_16  CLGITVWGVR DTDSWRSDQT PLLFGKKAAY AVLALNGGSE PP~~~~~~~~
             351                                                400
Z46264_1024  ~~~~~~~~~~ ~~~~~~~~ND ANEKGQRVGI ISWSDPTNNS WR~~~~~~~~ 
Z46945_1020  ~~~~~~~~~~ ~~~~~~~~ND ANEKGQRVGI ISWSDPTNNS WR~~~~~~~~ 
 U08894_341  SDAGTIKGVG SGRCLDVPNA STSDGVQLQL WDCHGGTNQQ WTYT~~~~~~ 
 U08894_369  ~~~~~~~~~~ ~~~~~~~~~A STSDGVQLQL WDCHGGTNQQ W~~~~~~~~~ 
 U08894_342  SDAGTIKGVG SGRCLDVPNA STSDGVQLQL WDCHGGTNQQ WTYTDSQELR 
    Stmxlna  ADGGQIKGVG SGRCLDVPDA STSDGTQLQL WDCHSGTNQQ WAATDAGELR 
 Z50866_840  ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~VRRAR HDGASGERRS WN~~~~~~~~ 
             401                                                450
 U08894_342  VYGNKCLDAA GTGNGTKVQI ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 
    Stmxlna  VYGDKCLDAA GTSNGSKVQI YSCWGGDNQK WRLNSDGSVV GVQSGLCLDA 
             451                        478
    Stmxlna  VGNGTANGTL IQLYTCSNGS NQRWTRT*