Homology Modeling Overview

As mentioned in the project description, our goal is to link five genes from the taxol biosynthesis pathway. In order to better understand the behavior of the proteins that we have isolated from the taxol biosynthesis pathway, we are using homology modeling to learn about active site architecture and catalytic functions. Homology modeling is based on the observation that related protein structures tend to have similar 3-D structures and functions. During homology modeling, 1 or more template proteins are used to identify structurally conserved regions, and to predict structurally variable regions that often include mutations from an already known structure. Through homology modeling, we can learn a lot about details about the protein such as active site architecture, ligand binding, and etc. Usually, when the target sequence is 30-50% similar (30%-50% identical amino acids) to the template sequence, they will share 80%+ shared 3-D structures. During the modeling process, we will be looking for template sequences with 30%+ sequence identity as good models for the target sequence.

Tools

PyMol: a software modeling application that allows users to view the 3-d structure of any proteins, including secondary, tertiary, and quaternary structures and the molecular interactions between side chains. It's the main tool that we use to visualize active site, and terminals of proteins.

ModWeb: a web-based database with all protein templates present and target sequences are matched with reliable models of template proteins.

Literature: previously published scholarly articles on research of proteins that include reliable template sequences for our specific model. Many mutations and active site construction has already been discovered in these articles.

badA

Template protein: 4RLQ
Organism: Rhodopseudomonas palustris (bacteria)
Class: PFAM00501 (ATP-dependent adenylation enzymes)
Plants and bacteria employ aroyl CoA thioesters for biosynthesis of specialized metabolites
Mechanism: ATP-dependent CoA ligases use 2 half-reactions to catalyze thioesterification

General Features
BadA has C-terminal/N-terminal domains joined by a flexible hinge

• badA always in conformation primed for thioesterification
• Nonconserved active site Lys427, present in badA active site in thiolation conformation, not required for adenylation but necessary for thioesterification

• N-terminal (1-434), C-terminal (435-522) domains fold analogously to the benzoate CoA ligase BCLM (2v7b)
• N-terminal contains benzoate binding site
• C-terminal domain contacts other edge of benzoate binding pocket, positions benzoate into active site through charged interaction between carboxylate of benzoate and Lys427

Features of active site
Largely hydrophobic
Para- carbon of BzO neighbors carbonyl of Leu332 along peptide backbone
2 meta- carbons of BzO point toward His333 & Ala227
Si- & re- faces of BzO positioned between backbone amide bonds, comprising Gly327, Ser328, Thr329 on 1 face, Tyr on other face
When Bz-AMP forms, Lys427 moves from interaction with carboxylate of substrate to several polar contacts with Bz-AMP & peptide backbone
Indicates Lys427 is needed for thiolation reaction

Rational mutation of the BadA active site
4 residues (Ala227, Leu332, His 333, Ile334) surround the phenyl ring of BzO in the active site
The residue positioned near a given carbon of BzO depends on whether BzO is in carboxylate-bound orientation or rotated Bz-AMP-bound orientation
Targeted mutations of Ala227Gly, Leu332Ala, His333Ala, Ile334Ala should show relative importance of 2 orientations

BadA structures & Homology Enzymes in this family typically fold in larger N-terminal domain (400-550 residues) & a smaller C-terminal domain (~110 residues), active site lies in between
N-terminal domain contains residues that bind carboxylate substrate
C-terminal residues coordinate ribose & phosphate groups
Phe226 residue in badA offset by ~72o away from BzO, opening CoA binding channel

Catalytically Important Lysine Residues Uses C-terminal lysine (Lys427/512 for BadA) to orient BzO substrate
Lys427 coordinates BzO in the active site & Lys512 is far from active site and solvent-exposed in C-domain
Lys427 of BadA makes 4 polar contacts with Bz-AMP, 1 with benzoyl oxygen, 3 with AMP moiety, and 2 to Gly303 & Gly430 (these contacts anchor Bz-AMP)
Predict Lys427Ala-badA mutant would show the second thioesterification of badA

• When badA assumes the adenylation conformation, Lys572 enters the active site