Functional discovery and promiscuity in the RuBisCO superfamily

Warlick, Benjamin

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https://hdl.handle.net/2142/45444 Copy

Description

Title

Functional discovery and promiscuity in the RuBisCO superfamily

Author(s)

Warlick, Benjamin

Issue Date

2013-08-22T16:40:21Z

Director of Research (if dissertation) or Advisor (if thesis)

Gerlt, John A.

Doctoral Committee Chair(s)

Gerlt, John A.

Committee Member(s)

• Cronan, John E.
• Nair, Satish K.
• Mitchell, Douglas A.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)
• RuBisCO-like proteins (RLPs)

Abstract

To keep pace with large scale sequencing projects that are filling sequence databases with misannotated sequences, a new method of functional annotation is necessary. The new method is called “genomic enzymology” and utilizes bioinformatics, molecular docking, microbiology, and enzymology. For a superfamily to benefit from this new strategy there must exist a set of solved functions and crystal structures. D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the carboxylation of D-ribulose 1,5-bisphosphate to yield two molecules of 3-phosphoglycerate. Currently, there are four forms of RuBisCO, three of which catalyze the canonical carboxylation reaction. Form IV RuBisCOs (also known as RuBisCO-like proteins (RLPs)) do not catalyze the carboxylation reaction and are of interest because they can provide a wealth of information on structure/function relationships of the superfamily. The work here focuses on identification of novel functions as well as determination of promiscuity in the (RuBisCO) superfamily. To identify novel functions, novel sequences were targeted for cloning, purification, and assaying. This approach was used on sequences in the family homologous to R. rubrum RLP that has a published function of 5-methylthio-D-ribulose 1-phosphate 1,3-isomerase. Efforts to isolate usable RLP failed, but collaborators at AECOM were able to purify functional enzyme of a downstream cupin that was then mechanistically characterized. The function of the cupin also provided evidence for the physiological context of the R. rubrum RLP. The investigation of promiscuity in the RuBisCO superfamily stems from previous research showing that an authentic RuBisCO can complement an RLP knockout. If an authentic RuBisCO were able to catalyze RLPs reactions, this would be an important element for determining the origin of RuBisCO and for determining the structure/function relationship. Both in vitro and in vivo analysis, showed no promiscuity in the authentic RuBisCO for RLP reactions.

Graduation Semester

2013-08

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http://hdl.handle.net/2142/45444

Copyright and License Information

Copyright 2013 Benjamin Warlick

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