DOI

10.17077/etd.aalpiyzu

Document Type

Dissertation

Date of Degree

Spring 2017

Access Restrictions

Access restricted until 07/13/2019

Degree Name

PhD (Doctor of Philosophy)

Degree In

Chemistry

First Advisor

Dey, Mishtu

First Committee Member

Kohen, Amnon

Second Committee Member

Quinn, Daniel M.

Third Committee Member

Gloer, James B.

Fourth Committee Member

Fuentes, Ernesto J.

Abstract

Cupins are a large superfamily of enzymatic and non-enzymatic members that contain a conserved β-barrel domain, or double-stranded β-helix (DSBH) fold. The cupin superfamily is one of the most functionally diverse groups of proteins known to exist. The vast majority of cupins contain a mononuclear metal binding site at the core of the DSBH fold capable of binding different metal ions. One of the largest cupin subfamilies is known as the Fe(II)/α-ketoglutarate (αKG)-dependent dioxygenases. Prolyl 4-hydroxylases (P4Hs) belong to the group of Fe(II)/αKG-dependent dioxygenases and catalyze the formation of 4R-hydroxyproline (Hyp) from various proline-containing substrates. The formation of Hyp is an important post-translational modification to many different proteins involved in essential biochemical pathways. Abnormalities in these pathways can lead to diseases such as cancer, fibrosis, respiratory issues, scurvy, and stroke. An Fe(II)/αKG-dependent prolyl hydroxylase from Bacillus anthracis (BaP4H) was investigated to understand its substrate recognition ability and catalytic properties. Novel crystal structures were solved that revealed conformational changes upon substrate binding and key interactions of various ligands in the active site for different catalytic steps. Although the majority of cupin family enzymes catalyze a reaction using iron as an essential cofactor, other metal cofactors can allow the diverse biological transformations carried out by this group of enzymes. A class of enzymes known as dimethylsulfoniopropionate (DMSP) lyases uses different metal ions to catalyze the formation of acrylate and dimethylsulfide (DMS) from DMSP. DMSP is one of the most prevalent and significant molecules to the life and biogeochemistry of the oceans. The products DMS and acrylate are environmentally significant and industrially valuable. DMSP is predominantly catabolized by marine bacteria and can serve different functions. One of the most abundant bacteria in the ocean, Pelagibacter, was determined to contain a DMSP lyase DddK. The DddK catalyzed DMSP lyase activity in the presence of different metal ions has shown that it catalytically prefers Ni(II) compared to other transition metal ions examined. Spectroscopic, site-directed mutagenesis, and crystallographic studies illustrate central residues responsible for metal ion binding and possible roles in transition state stabilization. A greater mechanistic understanding of DMSP lyases will lead to more impactful information about global environmental climate regulation.

Public Abstract

Enzymes exist in many different shapes and sizes, and facilitate certain chemical modifications to occur on a faster timescale than would normally happen. One of these shapes is similar to a sandwich and is referred to as a ‘cupin’ domain. Cupins typically contain a metal in the middle of the sandwich shape where different metals can cause altered outcomes, which is not well understood and makes cupin enzymes one of the most diverse enzyme families.

One enzymatic system that uses the cupin domain requires iron, oxygen, and an organic cofactor to make important modifications to proteins involved in different biological pathways. Many of these enzymes are present in humans and other organisms and contain similar structures to one another but are able to achieve distinct functions. If we are able to understand how this collection of enzymes modify each of its specific targets selectively, then designing better drugs and new antibiotics that generate fewer side effects and are more effective will be possible.

A different cupin enzyme family found in marine bacteria is responsible for assisting in the global sulfur cycle. These enzymes are able to use different metals present in the oceanic environment to catabolize an important and very abundant sulfur- containing molecule. This molecule gets broken down into two products significant for the environment and industry. Better insight into enzyme selectivity for active metals and how the sulfur-containing molecule gets broken down, could lead to important conclusions for other metalloproteins in addition to imperative environmental global climate knowledge.

Keywords

DMSP lyase, Prolyl 4-Hydroxylase

Pages

xxiii, 161 pages

Bibliography

Includes bibliographical references (pages 143-154).

Comments

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Copyright

Copyright © 2017 Nicholas Jay Schnicker

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