SRI International Bioinformatics 1 Orphan Enzymes Alexander Shearer, Tomer Altman, Anamika Kothari, Christian Ngo, Shahrzad Zarafshar

SRI International Bioinformatics 2 The problem – disconnected data

SRI International Bioinformatics 3 The problem – disconnected data

SRI International Bioinformatics 4 An orphan enzyme is an activity that has been extensively characterized in the lab…

SRI International Bioinformatics 5 …but for which no sequence is available in major databases

SRI International Bioinformatics 6 How many orphans? ResourceTotal sequencedTotal orphans Enzyme DB2,4611,783 UniProtKB/Swiss-Prot2,4621,782 UniProtKB/TrEMBL2,9491,295 BioCyc – Proteins 3,1021,142 BioCyc – Reactions 3,1191,125 Orenza3,1221,122 NCBI Psrotein3,1281,116 Final tally3,1281,116

SRI International Bioinformatics 7 Project goals Resolve as many orphans as possible Help others resolve orphans

SRI International Bioinformatics 8 How are orphans resolved? The sequence is out there! –Hidden in papers –Disconnected, in databases –“Sequence” is present in easy protein package… Never been sequenced –Purify and ID in the lab –May have been IDed for a different activity Dubious E.C. numbers…

SRI International Bioinformatics 9 Validating orphans

SRI International Bioinformatics 10 Validating orphans

SRI International Bioinformatics 11 Validating orphans

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SRI International Bioinformatics 15 Ranking orphans “Easy” ranking requires all of these: 1 – Purification protocol using standard affinity methods 2 – Source organism readily culturable 3 – Assay uses off-the-shelf substrates “Moderate” ranking requires any two of these: 1 – Purification protocol using standard affinity methods 2 – Source organism readily culturable 3 – Assay uses off-the-shelf substrates 4 – Known molecular weight Otherwise, it’s “Hard.” Activities can be bumped a level (e.g. “Moderate” to “Hard”) if the enzyme is labile, Protease sensitive, or otherwise hard to work with

SRI International Bioinformatics 16 How many true orphans? Resolved – 19% True Orphans – 81%

SRI International Bioinformatics 17 How do the rankings spread out? Hard – 35% Easy – 23% Moderate – 42%

SRI International Bioinformatics 18 Curious cases Perillyl alcohol Stereoisomers Cofactors ADH – when an instance is a class

SRI International Bioinformatics 19 Lab evaluation – ‘super-easy’ targets

SRI International Bioinformatics 20 Lab evaluation – general General method There is no unified lab identification process. The general plan is to use modern methods to update older protocols. For example, older protocols requiring multiple steps, gravity columns, difficult cell lysis methods, and swapping between dialysis tubing arrangements can be updated to use HPLC/FPLC, kit-based steps, and other modern tools. Similarly, older assays can be updated to use modern sensors, new specialty substrates, and, generally, methods that can use either a lower total concentration of the enzyme or a somewhat less purified form, helping to cut out purification time and costs. This example combination of Purification and Assay for is from a paper that was published over 40 years ago.

SRI International Bioinformatics 21 Future considerations What is a “good” annotation? Fixing issues with E.C. activities When does precision become overprecision? How do we find all the other misannotations? Other kinds of “PGDB-ready” orphans?

SRI International Bioinformatics 22 Catching NCBI misses…

SRI International Bioinformatics 23 Catching NCBI misses…