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4 Οκτ 2013 (πριν από 3 χρόνια και 8 μήνες)

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SRI International Bioinformatics

1

Orphan Enzymes


Alexander
Shearer,
Tomer

Altman,
Anamika

Kothari, Christian Ngo,
Shahrzad

Zarafshar

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The problem


disconnected data

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The problem


disconnected data

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An orphan enzyme is an activity that has been
extensively characterized in the lab…

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…but for which no sequence is available in
major databases

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How many orphans
?

Resource


Total sequenced

Total orphans


Enzyme DB


2,461


1,783


UniProtKB
/Swiss
-
Prot

2,462


1,782


UniProtKB/TrEMBL

2,949


1,295


BioCyc



Proteins

3,102


1,142


BioCyc



Reactions

3,119


1,125


Orenza


3,122


1,122


NCBI
Psrotein


3,128


1,116


Final tally


3,128


1,116

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Project goals


Resolve as many orphans as possible



Help others resolve orphans

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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…


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Validating orphans

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Validating orphans

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Validating orphans

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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



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How many true orphans?

Resolved


19%

True Orphans


81%

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How do the rankings spread out?

Hard


35%

Easy


23%

Moderate


42%

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Curious cases


Perillyl

alcohol


Stereoisomers


Cofactors


ADH


when an instance is a class

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Lab evaluation


‘super
-
easy’
targets

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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 1.1.99.9

is from a paper

that was published

over 40 years ago.

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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?