M-CSA Mechanism and Catalytic Site Atlas

Isotuberculosinol synthase

This enzyme is responsible for the conversion of tuberculosinyl diphosphate to tuberculosinol and the R and S iso-tuberculosinols. Tuberculosinols are one class of virulence factor in Mycobacterium tuberculosis. The active site for both of these functions is the same, but the R- and S-iso-tuberculosinol formation involves the addition of water at the C13 atom, rather than the C15, and involve an allylic rearrangement.

Reference Protein and Structure

Sequence: P9WJ61 (2.5.1.153) (Sequence Homologues) (PDB Homologues)
Biological species: Mycobacterium tuberculosis H37Rv (Bacteria)
PDB: 3wql - Crystal structure of Rv3378c with Mg2+ and PPi (2.1 Å)
Catalytic CATH Domains: 3.40.1180.10 (see all for 3wql)
Cofactors: Magnesium(2+) (1)

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Enzyme Reaction (EC:3.1.7.9)

water

CHEBI:15377

tuberculosinyl diphosphate(3-)

CHEBI:58822

→

diphosphate(3-)

CHEBI:33019

(13R)-edaxadiene

CHEBI:63178

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: Rv3378c,

Summary
Step 1
Step 2
Products
All Steps

Introduction

This mechanism represents formation of the 13R product. the Tyr51 abstracts a proton from the catalytic water, which attacks the C13 position. This initiates an allylic rearrangement and elimination of the phosphate group with concomitant deprotonation of Arg38.

Catalytic Residues Roles

UniProt	PDB* (3wql)
Asp34	Asp34B	Forms part of the magnesium binding site.	metal ligand
Tyr90	Tyr90B	No direct function in this mechanism proposal, although may help to activate the Arg38 to act as a general acid/base.	activator
Arg38, Tyr51	Arg38B, Tyr51B	Acts as a general acid/base.	proton acceptor, proton donor

*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic substitution with allylic rearrangement, proton transfer, overall reactant used, overall product formed, hydrolysis, inferred reaction step, native state of enzyme regenerated

References

Chan HC et al. (2014), J Am Chem Soc, 136, 2892-2896. Structure and inhibition of tuberculosinol synthase and decaprenyl diphosphate synthase from Mycobacterium tuberculosis. DOI:10.1021/ja413127v. PMID:24475925.
Nakano C et al. (2011), Biosci Biotechnol Biochem, 75, 75-81. Characterization of the Rv3378c gene product, a new diterpene synthase for producing tuberculosinol and (13R, S)-isotuberculosinol (nosyberkol), from the Mycobacterium tuberculosis H37Rv genome. DOI:10.1271/bbb.100570. PMID:21228491.

Step 1. Tyr51 abstracts a proton from the catalytic water, which attacks the C13 position. This initiates an allylic rearrangement and elimination of the phosphate group with concomitant deprotonation of Arg38.

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Catalytic Residues Roles

Residue	Roles
Asp34B	metal ligand
Tyr51B	proton acceptor
Arg38B	proton donor

Chemical Components

ingold: bimolecular nucleophilic substitution with allylic rearrangement, proton transfer, overall reactant used, overall product formed, hydrolysis

Step 2. Inferred return step. It is unknown if the transfer of a proton from Tyr51 to Arg38 is direct or mediated through one or more water molecules.

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Catalytic Residues Roles

Residue	Roles
Asp34B	metal ligand
Tyr90B	activator
Tyr51B	proton donor
Arg38B	proton acceptor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated

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Step 2. Inferred return step. It is unknown if the transfer of a proton from Tyr51 to Arg38 is direct or mediated through one or more water molecules.

Products.

Summary
Step 1
Step 2
Products
All Steps

Introduction

This mechanism represents formation of the 13S product. Tyr80 abstracts a proton from the catalytic water, which attacks the C13 position. This initiates an allylic rearrangement and elimination of the phosphate group with concomitant deprotonation of Arg38.

Catalytic Residues Roles

UniProt	PDB* (3wql)
Asp34	Asp34B	Forms part of the magnesium binding site.	metal ligand
Arg38, Tyr90	Arg38B, Tyr90B	Acts as a general acid/base.	proton acceptor, proton donor
Tyr51	Tyr51B	No direct function in this mechanism proposal.

Chemical Components

hydrolysis, overall product formed, overall reactant used, proton transfer, bimolecular nucleophilic substitution with allylic rearrangement, inferred reaction step, native state of enzyme regenerated

References

Chan HC et al. (2014), J Am Chem Soc, 136, 2892-2896. Structure and inhibition of tuberculosinol synthase and decaprenyl diphosphate synthase from Mycobacterium tuberculosis. DOI:10.1021/ja413127v. PMID:24475925.
Nakano C et al. (2011), Biosci Biotechnol Biochem, 75, 75-81. Characterization of the Rv3378c gene product, a new diterpene synthase for producing tuberculosinol and (13R, S)-isotuberculosinol (nosyberkol), from the Mycobacterium tuberculosis H37Rv genome. DOI:10.1271/bbb.100570. PMID:21228491.

Step 1. Tyr80 abstracts a proton from the catalytic water, which attacks the C13 position. This initiates an allylic rearrangement and elimination of the phosphate group with concomitant deprotonation of Arg38.

Download: Image, Marvin File