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Molecular markers of serine protease evolution

2001; Springer Nature; Volume: 20; Issue: 12 Linguagem: Inglês

10.1093/emboj/20.12.3036

1460-2075

Maxwell M. Krem,

Enzyme Structure and Function

Article15 June 2001free access Molecular markers of serine protease evolution Maxwell M. Krem Maxwell M. Krem Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, MO, 63110-1093 USA Search for more papers by this author Enrico Di Cera Corresponding Author Enrico Di Cera Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, MO, 63110-1093 USA Search for more papers by this author Maxwell M. Krem Maxwell M. Krem Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, MO, 63110-1093 USA Search for more papers by this author Enrico Di Cera Corresponding Author Enrico Di Cera Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, MO, 63110-1093 USA Search for more papers by this author Author Information Maxwell M. Krem1 and Enrico Di Cera 1 1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, MO, 63110-1093 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:3036-3045https://doi.org/10.1093/emboj/20.12.3036 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The evolutionary history of serine proteases can be accounted for by highly conserved amino acids that form crucial structural and chemical elements of the catalytic apparatus. These residues display non- random dichotomies in either amino acid choice or serine codon usage and serve as discrete markers for tracking changes in the active site environment and supporting structures. These markers categorize serine proteases of the chymotrypsin-like, subtilisin-like and α/β-hydrolase fold clans according to phylogenetic lineages, and indicate the relative ages and order of appearance of those lineages. A common theme among these three unrelated clans of serine proteases is the development or maintenance of a catalytic tetrad, the fourth member of which is a Ser or Cys whose side chain helps stabilize other residues of the standard catalytic triad. A genetic mechanism for mutation of conserved markers, domain duplication followed by gene splitting, is suggested by analysis of evolutionary markers from newly sequenced genes with multiple protease domains. Introduction Serine proteases carry out a diverse array of physiological and cellular functions, ranging from digestive and degradative processes to blood clotting, cellular and humoral immunity, fibrinolysis, fertilization, embryonic development, protein processing and tissue remodeling. Serine proteases have been classified into evolutionarily unrelated clans, which have been subdivided into families of proteases whose homology can be established statistically (Rawlings and Barrett, 1993; Barrett and Rawlings, 1995). Clans differ in terms of overall fold and the order of catalytic residues in the primary sequence. Despite these significant differences, serine proteases of clans SA (chymotrypsin-like) (Lesk and Fordham, 1996), SB (subtilisin-like) (Siezen and Leunissen, 1997) and SC (α/β-hydrolase fold) (Ollis et al., 1992) maintain a strictly conserved active site geometry among their catalytic Ser, His and Asp residues. This shared catalytic structure suggests that common architectural motifs are likely to be found in the molecular designs of active sites utilizing a Ser–His–Asp triad. Major steps in the assembly, improvement and specialization of the catalytic architecture should correspond to significant evolutionary transitions in the history of protease clans. Evolutionary markers encountered in the sequences contributing to the catalytic apparatus would thus give an account of the history of an enzyme family or clan and provide for comparative analysis with other families and clans. Therefore, the use of sequence markers associated with active site structure generates a model for protease evolution with broad applicability and potential for extension to other classes of enzymes. The first report of a sequence marker associated with active site chemistry was the observation that both AGY and TCN codons were used to encode active site serines in a variety of enzyme families (Brenner, 1988). Since AGY→TCN interconversion is an uncommon event, it was reasoned that enzymes within the same family utilizing different active site codons belonged to different lineages. This phenomenon was shown to apply to both the enzymes of clans SA and SC. Enzymes of clan SB have been reported to use exclusively TCN for the active site serine (Rawlings, 1998a). Despite the discovery of two exceptions to that rule in this study, the paucity of AGY active site codons in clan SB suggests that codon usage by that serine provides very limited evolutionary information. Investigation of additional markers associated with catalytic function would generate a more complete story. An example of a second marker associated with catalytic function in clan SA serine proteases is residue 225 (chymotrypsinogen numbering). Residue 225 has a Pro– Tyr dichotomy; proteases with Tyr225 are Na+-activated allosteric enzymes, displaying a regulation of catalytic activity not seen in enzymes with Pro225 (Dang and Di Cera, 1996). Several other highly conserved residues in serine protease families have been linked to catalysis, although they have not been explored as evolutionary markers. The backbone of Ser214 in chymotrypsin-like proteases contributes to the S1 binding pocket (Perona and Craik, 1995). The side chain of the same residue helps generate a polar environment for the catalytic Asp102 (McGrath et al., 1992), and both oxygens of Ser214 form hydrogen bonds with waters located in the active site cleft (Pletnev et al., 2000). Ser125 (subtilisin BPN′ numbering) in subtilisin-type proteases plays a similar role; its backbone contributes to the S1 binding pocket (Perona and Craik, 1995), whereas its side chain is directly adjacent to the catalytic residues (Siezen and Leunissen, 1997) so that the hydroxyl is within hydrogen-bonding distance of the catalytic Asp32. Several carboxypeptidases have a residue that functions similarly in the active site: the backbone nitrogen of unpaired Cys341 (yeast carboxypeptidase W numbering) hydrogen bonds to the side chain of catalytic Asp338 (Rudenko et al., 1995), while the side chain of Cys341 is proximal to the side chain of catalytic His397 (Shilton et al., 1997). Each of these residues (Ser214, Ser125 and Cys341) appears to be the fourth member of a catalytic tetrad in its respective protease clan. We examined the above residues and found that they utilized dichotomous sequence choices. We also analyzed the sequences of proteases from clans SA, SB and SC to identify other conserved residues potentially related to active site structure or function. Absolutely conserved non-serine residues were avoided, as they were likely to yield little evolutionary information. We identified two residues (Ser190 and Ser207) in clan SB and one residue (Ser57) in clan SC that fit the conservation and dichotomy criteria for discrete evolutionary markers. Our analysis proceeded in two steps: division of the clans into families or subfamilies based on primary structure motifs surrounding the active site residues and non-active site evolutionary markers, and categorization of each family according to single-residue evolutionary markers. The results reveal the governing role of the active site in protease evolution and indicate that domain duplication followed by gene splitting may be responsible for the generation of new evolutionary lineages. Results Markers divide clans into families Protease clans SA, SB and SC have been subdivided into different families (referred to as ‘subfamilies’ within the subtilisin ‘superfamily’) whose homology has been established by sequence (Rawlings and Barrett, 1993; Barrett and Rawlings, 1995). Such distinctions are based on the sequence of complete protease domains. The present study finds that the above distinctions can be recapitulated using short primary sequence elements surrounding the active site residues and other highly conserved residues that have been identified as evolutionary markers by the present study (Table I). This result indicates that alterations in active site structure accompanied the divergence of the various families within clans SB and SC. Thus, significant functional differences map to changes in narrowly defined regions of the molecule, especially within the active site. Table 1. Sequence motifs surrounding evolutionary markers and active site residues for major families/subfamilies of clans SA, SB and SC SA family His57* Asp102* Ser195* Ser214 Pro/Tyr225 S1 T A A H C D I A L G D S G G P G I V S W P/Y G V Y/F SB subfamily Asp32* His64* Ser125 Ser190 Ser207 Ser221* S8A D T G I G H G T H N M S L G G F S N Y I L S T G T S M A T S8B D D G I K/R H G T R S A S W G P Y S E X I X T T G T S A S A S8C D T G I G H G T H N/S X S L G X F/W S S R I L S X G T S M A T/S SC family Ser57 Ser146* Asp338* and Val/Cys341 His397* S9 G G P G – S X G W S Y G G G X X D X N V/C G A G H S10 G G P G C S S G E S Y A G G D X D X X V/C D E X H Conserved and active site residues are in bold. Active site residues are marked with an asterisk (). ‘X’ indicates significant variation for a particular residue. Markers divide families into lineages Within families, sequence changes occurring at active site and marker locations are subtler. These sequence changes can be traced through residues that feature usage of two dissimilar amino acids, such as Pro/Tyr225 in clan SA and Val/Cys341 in clan SC. Similar alterations can be observed in the silent but non-conservative difference between usage of TCN or AGY codons for highly conserved serines, such as Ser195 and Ser214 in clan SA; Ser125, Ser190 and Ser207 in clan SB; and Ser57 and Ser146 in clan SC. The above sequence differences represent significant mutations, and chronicle changes in and around the active site. Such changes, all of which require double-nucleotide shifts, would be extremely rare. Accordingly, they represent the best candidates for serving as discrete evolutionary markers that subdivide protease families into distinct lineages. The protease domains analyzed in this study are categorized according to evolutionary markers in Table II. Table 2. Categories of serine proteases according to evolutionary markers A Ser214:TCN Ser214:AGY Ser195:TCN Pro225 Chymotrypsin (vertebrates, invertebrates); chymotrypsin-like (human); easter (fly); enterokinase (mammals); granzymes (mammals)a; kallikrein—tissue and glandular (mammals); trypsin (vertebrates, invertebrates) elastase (mammals); factor Be (mammals); factor C2 (mammals)e; factor XI (human); factor XII (mammals); HGF (mammals)f; HGFA (human); kallikrein—plasma (mammals); Sp14D1 (mosquito); t-PA (mammals) Ser195:TCN Tyr225 MASP-1 (mammals); gastrulation defective (fly) factor B (sea urchin); haptoglobin (mammals)g; nudel (fly) Ser195:AGY Pro225 apolipoprotein(a) (mammals)b; CG-18735 (fly); masqueradec (fly); neurotrypsin (mammals); plasmin (mammals) acrosin (mammals); hepsin (mammals) Ser195:AGY Tyr225 CASP (hamster); cercarial protease (schistosome)d; factor C1r (human); factor C1s (human); MASP-2 (human); thrombin (vertebrates) factor VII (mammals)h; factor IX (mammals); factor X (mammals, birds); protein C (mammals) B Ser214:TCN Ser214:AGY Ser195:TCN Pro225 degradative, development, cell-mediated immunity, kallikrein cell-mediated immunity, clotting, degradative, fibrinolytic/HGFA, HGF, humoral immunity Ser195:TCN Tyr225 humoral immunity, development development, humoral immunity Ser195:AGY Pro225 degradative, development, fibrinolytic degradative Ser195:AGY Tyr225 clotting, humoral immunity clotting C Ser190:TCN Ser190:AGY Ser125:TCN Ser207:TCN S8A: intracellular protease I (Bacillus subtilis); proteinase K (Tritirachium album); sexual differentiation serine protease (Schizosaccharomyces pombe); subtilisin 1 (B.subtilis); subtilisin-like protease III (Saccharomyces cerevisiae) S8A: alkaline protease (Acremonium chrysogenum); alkaline protease (Trichoderma harzianum)l; serine protease (Paenibacillus polymyxa) S8C: cucumisin (melon)i; cucumisin-like AF036960 (soybean)i; TagB,C (Dictyostelium discoides); TMP (tomato)i S8C: cucumisin-like ARA12 (A.thaliana)i; cucumisin-like sbt1 (tomato)i Ser125:TCN Ser207:AGY S8A: minor extracellular serine protease vpr (B.subtilis) S8B: blisterase 4 (C.elegans)j,k; celfur (C.elegans)k; kex2-like endoprotease 1 (fly); kexin 2 (Candida albicans)l S8B: amon (fly)k,p; kexin 1 (S.pombe)k; kexin 2 (h.crab)k S8C: TPP-II-like F21H12.3 (C.elegans)q,r Ser125:AGY Ser207:TCN S8A: aqualysin I (Thermus aquaticus); C5a peptidase (Streptococcus pyogenes); subtilisin-like (Plasmodium berghei) S8C: SKI-1 (mammals); TPP-II (mammals); TPP-II-like T13K14.10 (Arabidopsis thaliana)m S8C: TPP-II-like SPAP8A3.12c (S.pombe)r,s; TPP-II (fly)l Ser125:AGY Ser207:AGY S8A: alkaline protease (B.amyloliquifaciens); cell wall protease (B.subtilis); elastase (Aspergillus flavus); epidermin (Staphylococcus epidermidis); subtilisin carlsberg (Bacillus licheniformis); subtilisin E (B.subtilis); S8A: nisin operon protease (Lactococcus lactis)k S8B: furin (Aplysia californica)k,n; PACE4 (mammals)j,k; PC1 (mammals)o; PC5 (mammals, lancelet)j,k S8B: calcium-dependent protease (A.variabilis)k; PC2 (mammals)k,p; PC3 (mammals, frog)k; PC3-like (hydra)k,l,t D Ser146:TCN Ser146:AGY Ser57:TCN Val341 S9: APEH-like (Thermoplasma acidophilum)u; APEH (mammals); APEH-like (A.thaliana); DPP-B (S.cerevisiae); ome (fly) S9: APEH-like (C.elegans)u; DPP (S.pombe); oligopeptidase B (Escherichia coli)u S10: CG-4572 (fly); carboxypeptidase I (A.thaliana, barley, rice, tomato); glucose acyltransferase (potatov, tomato); vitellogenic carboxypeptidase (human) S10: BG: DS00365.3(a) (fly); carboxypeptidase D (barley, ricev); carboxypeptidase D-like AP002539 (rice); carboxypeptidase II (rice)v Ser57:TCN Cys341 S10: carboxypeptidase A (C.elegans); carboxypeptidase Y (C.albicans); vitellogenic carboxypeptidase (mosquito) S9: APEH-like orf-c01017 (Sulfolobus solfataricus)ab; APEH-like APE1547 (A.pernix)ab S10: BG:DS00365.3(b) (fly)u; CG-3344 (fly); carboxypeptidase D (S.cerevisiae) Ser57:AGY Val341 S9: allergen (Trichophyton rubrum)w,x; APEH-like APE2441 (Aeropyrum pernix); DPP-IV (vertebrates); FAP (mammals); prolyl oligopeptidase (mammals)y S9: alanyl DPP (Aspergillus oryzaex, Xylella fastidiosav,w); DPP-VI (mammals)v,ac S10: virulence protein NF314 (Naegleria fowleri) Ser57:AGY Cys341 S9: APEH-like PH0863 (Pyrococcus horikoshii)z; APEH-like SC66t3.21c (Streptomyces coelicolor)aa S9: APEH-like PAB1418 (Pyrococcus abyssi)w; APEH-like YuxL (B.subtilis)ad; APEH-like DR0165 (Deinococcus radiodurans)z S10: carboxypeptidase Y (A.thaliana); carboxypeptidase-like (Matricaria chamomilla) S10: carboxypeptidase A (mammals) A, clan SA category assignments. B, functional assignments of clan SA marker lineages. C, clan SB category assignments. D, clan SC category assignments. Gene accession numbers are provided for newly sequenced genes of undetermined function. Letters in parentheses following gene names indicate individual protease domains from multi-protease genes; the alphabetical order indicates the 5′→3′ sequence of the domains within the gene. Annotations are provided for those enzymes whose category title does not precisely match their amino acid or codon usage; such exceptions are placed in the categories that most closely correspond to the enzymes' amino acid or codon usage. a Mouse granzyme F, Thr214; rat granzyme II, Ser214:AGT; b rhesus monkey, Asn195; c Gly195; d Ile225; e insertion after residue 224; f Tyr195, Val214; g Ala195; h mouse, Ile225; rabbit, Val225; i Ala207; j Leu190; k Thr207; l Thr190; m Pro207; n Thr125; o Ala190; p Asp190; q Gly207; r Ser221:AGT; s Cys190; t Cys125; u Ala57; v Ile341; w Gly57; x Leu341; y Asn57; z Thr57; aa Trp57; ab Thr341; ac Asp146; ad Met57. APEH, acyl-peptide hydrolase; CASP, calcium-activated serine protease; DPP, dipeptidyl peptidase; FAP, fibroblast activation protein; HGF, hepatocyte growth factor; HGFA, hepatocyte growth factor activator; MASP, mannose-associated lectin-binding serine protease; PACE, paired basic amino acid cleaving enzyme; PC, prohormone convertase; SKI, subtilisin/kexin isozyme; Sp, serine protease; Tag, tight aggregate stage; TMP, tomato meiotic proteinase; t-PA, tissue plasminogen activator; TPP, tripeptidyl peptidase. CG- and BG- prefixes refer to Drosophila genes sequenced by Celera Genomics and the Berkeley Drosophila Genome Project, respectively. This table provides a limited sample of sequences from each protease clan. A complete listing of the sequences analyzed and categorized is available as Supplementary data (at The EMBO Journal Online). As each peptidase clan has been assigned three binary evolutionary markers, each clan is subdivided into eight groups. The segregation is not random. Enzymes that have high sequence similarity tend to group into the same lineage. Different copies of enzymes from the same organism or closely related organisms are almost always categorized within the same lineage. For example, the enzymes considered to be part of the plasminogen activator/hepatocyte growth factor activator subfamily of serine proteases (Miyazawa et al., 1998) are all part of the Ser195:TCN/Ser214:AGY/Pro225 lineage in clan SA. The mammalian enzymes of the prohormone and proprotein convertase subfamily (Seidah et al., 1994) are found exclusively within two lineages, Ser125:AGY/Ser190: TCN/Ser207:AGY and Ser125:AGY/Ser190:AGY/Ser207: AGY, of clan SB. Occasionally, proteases with low sequence similarity are found in the same group, as with the schistosomal cercarial protease and thrombin in lineage Ser195:AGY/Ser214:TCN/Tyr225. The low mutation rate of the binary markers suggests common ancestry for proteases such as the cercarial protease and thrombin, sequences that might otherwise be considered unrelated. Clan SA is unique among the three clans studied in that nearly all of its enzymes are extracellular. As a result, proteases of that clan have been shown to participate in a wide variety of physiological functions. Functions map roughly to the categories, with individual functions mapping to between one and four categories (Table IIB). In cases where particular functions map to multiple categories, these categories differ by one or two binary markers in the majority of cases. Categorizations that place enzymes associated with a particular function in the same category are in agreement with phylogenetic information based on extensive or complete sequence comparisons indicating a common lineage. Categorizations that place enzymes associated with a particular function (such as clotting) in significantly different categories are suggestive of different lineages giving rise to the enzymes of that function (e.g. factors X and XI in the clotting system) and also fit previous phylogenetic analyses (Krem et al., 1999). Enzymes of the complement and vitamin K-dependent clotting systems map to the same categories, but they do not map to the same categories as enzymes of fibrinolysis, cell-mediated immunity and tissue degradation (which includes digestion). This result concurs with previous studies indicating different lineages for the clotting and fibrinolytic cascades (Patthy, 1985, 1990). Thus, the separation of chymotrypsin-like proteases into discrete lineages agrees with previous partitioning of serine proteases and duplicates recognizable trends, doing so with minimal information. All three markers contribute information, as elimination of any one of the markers would place sequences that have been previously documented as having limited similarity in the same categories and impair the already limited functional resolution of the method. For example, elimination of the Pro/Tyr225 marker would merge the predominantly degradative category of Ser195:AGY/Ser214:TCN/Pro225 with the predominantly complement category of Ser195:AGY/Ser214: TCN/Tyr225. While the use of binary markers appears less suited to accurate functional prediction than sequence comparison and dendrogram construction, changes in the active site environment appear to have contributed significantly to functional radiation within clan SA. In clan SB, the clearest example of the segregative power of a binary marker is the exclusive use of categories with Ser207:AGY (including Thr207:ACN) by subfamily S8B; no enzymes in the kexin subfamily (S8B) utilize TCN at residue 207. Thus, a codon switch at residue 207 occurred with the emergence of the kexin subfamily. The kexin subfamily appears to be further subdivided by codon usage at Ser125. With the exception of the calcium-dependent protease of Anabaena variabilis, Ser125:AGY is found exclusively in metazoans. Some metazoans, yeast and fungi use Ser125:TCN. Mammalian sequences from subfamily S8B—and also subfamily S8C—only use Ser125:AGY. In subfamily S8A, the most highly conserved codon choice is found at residue Ser190. Thirty-nine of 45 sequences surveyed utilized TCN. Five of the six sequences utilizing Ser190:AGY mapped to the same lineage. As with clan SA, all three markers chosen for clan SB contain evolutionary information. The pattern of category occupation in clan SB further suggests that the various lineages of each subfamily of the subtilisin clan diverged subsequently to the division of clan SB into subfamilies. In clan SC, families S9 and S10 differ in category occupation, although not as dramatically as the subfamilies of clan SB. Nevertheless, it appears that the individual lineages within families radiated after the evolution of individual families within clan SC. Within family S10, plant and animal/fungal carboxypeptidases tend to occupy different lineages. Plant sequences dominate categories Ser57:TCN/Ser146:AGY/Val341 and Ser57:AGY/Ser146: TCN/Cys341; animal and fungal carboxypeptidases dominate categories Ser57:TCN/Ser146:TCN/Cys341, Ser57: TCN/Ser146:AGY/Cys341 and Ser57:AGY/Ser146:AGY/Cys341. Within family S9, the major species divide resides within the Val/Cys341 marker. No eukaryotic sequences have Cys341; that distinction belongs to acyl peptide hydrolase-like enzymes from a variety of thermophilic bacteria. The resulting paradox is that in family S9 the category Ser57:AGY/Ser146:AGY/Cys341 is occupied solely by thermophilic bacteria, while in family S10 the very same category is occupied solely by mammalian lysosomal carboxypeptidases. The utilization of Cys341 in family S9 appears to be a development unique to thermophilic bacteria. Construction of phylogenies based on markers Discrete evolutionary markers lend themselves to the construction of phylogenies. Phylogenies in this study were based on the presumption that TCN, Pro and Val are primordial compared with AGY, Tyr and Cys at the residues serving as markers. Evidence for the primordiality of TCN comes from previous sequence analyses comparing conserved and non-conserved serine residues. Diaz-Lazcoz et al. (1995) examined several classes of enzymes employing serine nucleophiles, including serine proteases, and demonstrated that catalytic serine residues utilized TCN codons to a greater degree than non-conserved serine residues. For serine proteases, the greater preference for TCN at active site residues was found to be statistically significant, with P(χ2) = 0.04. Because highly conserved residues are more likely than non-conserved residues to retain their original codon usage, it was concluded that TCN codons were the primordial serine codons. Comparative analysis of mammalian and fruit fly chymotrypsin-like sequences at residue 225 also agrees with functional evidence (Guinto et al., 1999) of the primordiality of Pro at that position. The fruit fly has Pro225 91% of the time compared with 82% for mammals, with P(χ2) <0.05. The case for the primordiality of Val as opposed to Cys at residue 341 of clan SC is implied by the preponderance of Val in families S9 [76% Val, n = 45, P(χ2) <0.01] and S10 [67% Val, n = 64, P(χ2) <0.01]. Perhaps the most convincing argument for the modernity of AGY, Tyr and Cys at the positions described above is that the enzymes utilizing those choices tend to be from higher metazoans and perform functions associated with a high degree of physiological complexity, such as blood clotting and prohormone processing. Figure 1 depicts phylogenies that are rooted based upon the primordiality of TCN, Pro and Val; despite this fact, the topology is invariant. Transitions involving two or three markers at one time were presumed to be unlikely. To identify the more likely single-marker transitions, we searched for individual enzymes that have close sequence relationships to enzymes in more modern categories. The protein–protein distances between each enzyme in a potential parent group (‘parent enzyme’) and the enzymes in the potential daughter group were averaged. Those smallest average distance values were used to choose the likely parent groups and enzymes for the daughter groups. This method allows the identification of the parent enzyme, which is similar to the ancestral enzyme that would have given rise to the daughter group (Figure 1). A small number of the pathways selected are not statistically superior to their alternatives. This raises the possibility that enzymes in some categories may have reached common evolutionary ‘destinations’ by following different pathways. Large amounts of variation within certain lineages also inflate the standard deviations of several average distances. In cases of numerical ties, the average distances between all enzymes in the parent and daughter groups were calculated and used to select a pathway. Figure 1.Phylogenetic category-based evolutionary pathways of serine proteases. More likely evolutionary transitions are shown with arrows; dashed lines indicate less likely transitions. Numbers indicate average distances ± standard deviations of the closest enzyme in each potential parent group to the enzymes in the potential daughter group(s). Numbers in parentheses indicate average distances ± standard deviations of all enzymes in each potential parent group to the enzymes in the potential daughter groups(s). Names of ‘parent enzymes’ and their species of origin are listed beneath average distances. Beneath names of parent enzymes are the probability values that pathway choices are the result of a random distance distribution. Probability values in parentheses correspond to average distance values in parentheses. Distances without standard deviations indicate daughter groups that contain only one enzyme. Download figure Download PowerPoint The phylogeny of family S1 of clan SA yields a new perspective of the evolution of that family. Trypsin is the selected parent enzyme for the two largest daughter groups emerging from the Ser195:TCN/Ser214:TCN/Pro225 lineage. Those two daughter groups, Ser195:AGY/Ser214: TCN/Pro225 and Ser195:TCN/Ser214:AGY/Pro225, contain a variety of enzymes that perform both degradative and more advanced physiological functions such as fibrinolysis and embryonic development. The enzymes in the daughter categories could therefore be viewed as more specialized relatives of the degradative enzymes like trypsin. The above daughter categories give rise to their own daughter categories, which show further enzyme specialization. The lineage Ser195:TCN/Ser214:AGY/Pro225 gives rise to Ser195:TCN/Ser214:AGY/Tyr225 through the parent enzyme Sp14D1. The daughter lineage contains enzymes involved in immunity (haptoglobin and sea urchin factor B) and development (fruit fly nudel); Sp14D1 is a hemolymph protein that has been implicated in the mosquito immune response (Gorman et al., 2000). The lineage Ser195:AGY/Ser214:TCN/Pro225 gives rise to Ser195:AGY/Ser214:TCN/Tyr225 through the uncharacterized fly gene CG-