Pages 1–5 1gp4 Evolutionary trace report by report maker August 20, 2009 4.3.3 DSSP 5 4.3.4 HSSP 5 4.3.5 LaTex 5 4.3.6 Muscle 5 4.3.7 Pymol 5 4.4 Note about ET Viewer 5 4.5 Citing this work 5 4.6 About report maker 5 4.7 Attachments 5 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1gp4): Title: Anthocyanidin synthase from arabidopsis thaliana (selenome- thionine substituted) Compound: Mol id: 1; molecule: anthocyanidin synthase; chain: a; synonym: leucoanthocyanidin dioxygenase; engineered: yes Organism, scientific name: Arabidopsis Thaliana; 1gp4 contains a single unique chain 1gp4A (346 residues long). 2 CHAIN 1GP4A 2.1 Q96323 overview CONTENTS From SwissProt, id Q96323, 94% identical to 1gp4A: 1 Introduction 1 Description: Leucoanthocyanidin dioxygenase (EC 1.14.11.19) (LDOX) (Leucocyanidin oxygenase) (Leucoanthocyanidin hydro- 2 Chain 1gp4A 1 xylase) (Anthocyanidin synthase) (ANS). 2.1 Q96323 overview 1 Organism, scientific name: Arabidopsis thaliana (Mouse-ear cress). 2.2 Multiple sequence alignment for 1gp4A 1 Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 2.3 Residue ranking in 1gp4A 1 Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 2.4 Top ranking residues in 1gp4A and their position on eudicotyledons; rosids; eurosids II; Brassicales; Brassicaceae; Ara- the structure 1 bidopsis. 2.4.1 Clustering of residues at 25% coverage. 2 Function: Oxidation of leucoanthocyanidins into anthocyanidins. 2.4.2 Overlap with known functional surfaces at Catalytic activity: Leucocyanidin + 2-oxoglutarate + O(2) = cis- and 25% coverage. 2 trans-dihydroquercetins + succinate + CO(2). Cofactor: Binds 1 iron ion and 1 ascorbate molecule per subunit. 3 Notes on using trace results 3 Pathway: Flavonoid synthesis; anthocyanidins biosynthesis. 3.1 Coverage 3 Similarity: Belongs to the iron/ascorbate-dependent oxidoreductase 3.2 Known substitutions 4 family. 3.3 Surface 4 About: This Swiss-Prot entry is copyright. It is produced through a 3.4 Number of contacts 4 collaboration between the Swiss Institute of Bioinformatics and the 3.5 Annotation 4 EMBL outstation - the European Bioinformatics Institute. There are 3.6 Mutation suggestions 4 no restrictions on its use as long as its content is in no way modified and this statement is not removed. 4 Appendix 4 4.1 File formats 4 2.2 Multiple sequence alignment for 1gp4A 4.2 Color schemes used 4 For the chain 1gp4A, the alignment 1gp4A.msf (attached) with 611 4.3 Credits 5 sequences was used. The alignment was downloaded from the HSSP 4.3.1 Alistat 5 database, and fragments shorter than 75% of the query as well as 4.3.2 CE 5 duplicate sequences were removed. It can be found in the attachment 1 Lichtarge lab 2006 2.4 Top ranking residues in 1gp4A and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 3 shows residues in 1gp4A colored by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Fig. 1. Residues 2-174 in 1gp4A colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.) Fig. 2. Residues 175-347 in 1gp4A colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.) to this report, under the name of 1gp4A.msf. Its statistics, from the alistat program are the following: Fig. 3. Residues in 1gp4A, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 611 Total number of residues: 191106 Smallest: 260 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Largest: 346 top 25% of all residues, this time colored according to clusters they Average length: 312.8 belong to. The clusters in Fig.4 are composed of the residues listed Alignment length: 346 in Table 1. Average identity: 34% Table 1. Most related pair: 99% cluster size member Most unrelated pair: 14% color residues Most distant seq: 32% red 86 48,51,73,74,77,78,79,80,81 84,85,86,87,102,103,105,106 110,138,139,141,172,179,187 Furthermore, <1% of residues show as conserved in this ali- 188,190,191,192,193,198,213 gnment. 215,216,217,218,220,221,223 The alignment consists of 48% eukaryotic ( 48% plantae) 225,226,227,228,231,232,233 sequences. (Descriptions of some sequences were not readily availa- 234,238,239,241,242,243,244 ble.) The file containing the sequence descriptions can be found in 246,248,249,250,251,257,260 the attachment, under the name 1gp4A.descr. 262,266,267,268,269,270,272 273,274,276,277,279,280,281 2.3 Residue ranking in 1gp4A 283,285,286,288,289,290,292 293,298,300,302,304,307 The 1gp4A sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues continued in next column in 1gp4A can be found in the file called 1gp4A.ranks sorted in the attachment. 2 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 300 S S(96)C 0.06 10/1 2.77 T(1).VY N 215 N S(23)Y 0.07 17/0 3.18 site N(64) H(4)Q T(3) A(2)LIC EXRV 241 I L(85) 0.09 6/0 4.17 I(11) V(2)F.Q M 304 F F(92) 0.11 9/0 3.30 L(4)VT .(1)IAC SGYPH 213 K K(42) 0.14 4/0 4.31 R(27) Fig. 4. Residues in 1gp4A, colored according to the cluster they belong to: V(1) red, followed by blue and yellow are the largest clusters (see Appendix for .(14)I the coloring scheme). Clockwise: front, back, top and bottom views. The Q(2) corresponding Pymol script is attached. A(3) N(1) L(1)GES Table 1. continued T(1) cluster size member H(1)F color residues 302 A A(78) 0.18 2/0 3.62 P(11)T Table 1. Clusters of top ranking residues in 1gp4A. V(4) G(2)SL. YD 2.4.2 Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier Table 2. and the heteroatom name used in that file. The top 25% of residues in 1gp4A at the interface with 2- oxyglutaric acid.(Field names: res: residue number in the PDB entry; type: 2-oxyglutaric acid binding site. Table 2 lists the top 25% of resi- amino acid type; substs: substitutions seen in the alignment; with the percen- dues at the interface with 1gp4AAKG370 (2-oxyglutaric acid). The tage of each type in the bracket; noc/bb: number of contacts with the ligand, following table (Table 3) suggests possible disruptive replacements with the number of contacts realized through backbone atoms given in the for these residues (see Section 3.6). bracket; dist: distance of closest apporach to the ligand. ) Table 2. res type subst’s cvg noc/ dist antn Table 3. (%) bb (A˚ ) res type disruptive 234 D D(99).E 0.00 5/0 3.77 site mutations 249 L L(99)T. 0.01 6/1 4.14 234 D (R)(FW)(H)(VCAG) F 249 L (R)(Y)(KH)(TE) 232 H H(99)T. 0.02 11/0 3.25 site 232 H (E)(MD)(Q)(TLPI) R 217 Y (K)(R)(Q)(E) 217 Y Y(98)LM 0.03 13/0 2.83 site 288 H (E)(T)(D)(M) FQ 298 R (D)(TY)(E)(S) 288 H H(98)K. 0.04 6/0 3.54 site 300 S (KR)(M)(QH)(FW) ASLQ 215 N (Y)(H)(FW)(R) 298 R R(98).X 0.06 9/0 2.82 site 241 I (Y)(R)(T)(H) AGIM continued in next column continued in next column 3 Table 3. continued 3.2 Known substitutions res type disruptive One of the table columns is “substitutions” - other amino acid types mutations seen at the same position in the alignment. These amino acid types 304 F (K)(E)(Q)(R) may be interchangeable at that position in the protein, so if one wants 213 K (Y)(FW)(T)(CG) to affect the protein by a point mutation, they should be avoided. For 302 A (R)(K)(E)(Y) example if the substitutions are “RVK” and the original protein has an R at that position, it is advisable to try anything, but RVK. Conver- Table 3. List of disruptive mutations for the top 25% of residues in sely, when looking for substitutions which will not affect the protein, 1gp4A, that are at the interface with 2-oxyglutaric acid. one may try replacing, R with K, or (perhaps more surprisingly), with V. The percentage of times the substitution appears in the alignment is given in the immediately following bracket. No percentage is given in the cases when it is smaller than 1%. This is meant to be a rough guide - due to rounding errors these percentages often do not add up to 100%. 3.3 Surface To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by 2 at least 10A˚ , which is roughly the area needed for one water mole- cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms.