Proteopedia - User contributions [en]

Bacterial thiolase

2010-05-21T15:43:54Z

Satyan Sharma: New page: '''3D structure (1DM3) of the bacterial ''Zoogloea ramigera'' biosynthetic thiolase''' == Introduction == Thiolase is best known as a key enzyme in the fatty acid degradation pathway, a...

'''3D structure (1DM3) of the bacterial ''Zoogloea ramigera'' biosynthetic thiolase'''

== Introduction ==

Thiolase is best known as a key enzyme in the fatty acid degradation
pathway, also known as the β-oxidation pathway. However, in the human
metabolism at least six thiolases (Table 1) have been
characterized. Each of them important in different pathways, and in
different cell organelles. Some thiolases are dimers, some thiolases are
tetramers, being dimers of dimers. Thiolases are involved, either in
degradative pathways (E.C. 2.3.1.16), or in biosynthetic pathways
(E.C. 2.3.1.9). This division is somewhat arbitrary as all thiolases
are sequence related to each other and catalyse the reaction in both directions.

[[Image:Fig1.png|700px]]

The above scheme shows the degradative reaction catalysed by thiolase. In this the acetoacetyl-CoA is degraded with CoA as cosubstrate, by which two molecules of acetyl-CoA are formed. Each thiolase catalyzes this reaction in both directions, but the
equilibrium is far much favoring the degradative direction
(Keq is 105, favoring the degradation). The
reaction is catalyzed by the enzyme as a two step reaction. In the first step a fully conserved
active site cysteine, Cys89 is acetylated, liberating acetyl-CoA. In
the second step the acetyl-moiety is transferred to CoA.

The best studied thiolase is the bacterial ''Zoogloea ramigera'' thiolase,
which is a biosynthetic thiolase and which catalyzes the formation of
acetoacetyl-CoA from two molecules of acetyl-CoA. The enzymological
properties of ''Zoogloea ramigera'' thiolase have been extensively
characterized by C.T Walsh and coworkers<ref>PMID:1353760</ref> and more recently its
structural enzymological properties have been characterized also.<ref
name="kursula">PMID:12501183</ref><ref
name="merilainen">PMID:19842716</ref> The residue numbering on this page refers to the ''Zoogloea ramigera''
thiolase sequence, and the structural information concerns the 1DM3 structure.

[[Image:Reaction cycle.png|right|thumb|300px|Figure 1. The catalytic cycle of thiolase. The synthetic direction is the clockwise direction. In this direction first Cys89 is acetylated by acetyl-CoA. In the second step this acetyl group is transferred to acetyl-CoA, the Claisen condensation.]]

{| class="wikitable" style="text-align:center
|+ Table 1. The six identified human thiolases.
! width="125"|Intracellular localization
! width="125"|Mode of assembly
!width="125"|Function
!width="325"|Comment: thiolase protein code (gene sequence code)

|-
| cytosol
| α4 || synthesis || CT (THIC)
|-
| mitochondria
| α4 || synthesis and degradation || T2 (THIL) Many point mutation variants related to metabolic disorders have been characterized.<ref name="Fukao">PMID:20156697</ref>

|-
|
| α4 || degradation || T1 (THIM)
|-
|
| α4β4 || degradation || trifunctional enzyme, TFE (ECHB)
|-
| peroxisome
| α2 || degradation || A/B (THIK)
|-
|
| α2 || degradation || SCP2 (NLTP)
|}

== The reaction mechanism ==

[[Image:Fig3 tetramer.png|left|thumb|300px|Figure 2: the tetramer (1DM3)]]

The complete catalytic cycle of the ''Zoogloea ramigera'' thiolase is
shown in Figure 1. This thiolase is a tetramer, indeed a dimer of
dimers, as shown in Figure 2. The active site is deeply
buried. It is constructed by <scene name='User:Satyan_Sharma/Sandbox_1/Acsite_1dm3/2'>four loops</scene>, which provide
the catalytic nucleophile (Cys89), the base (Cys378), as well as
Asn316 and His348. Asn316 and His348 are key residues of the active
site geometry: a Asn316-Wat82 dyad, together with His348 make <scene name='User:Satyan_Sharma/Sandbox_1/Oxyanionhole_1/2'>oxyanion hole 1</scene>, stabilizing the CoA-thioester enolate intermediate. The hydrogen bond donors of oxyanion hole 1 are Wat82 and NE2 (His348). This enolate is formed from acetyl-CoA after proton
abstraction by the catalytic base, Cys378 (Figure 3).
<applet load='1dm3' size='300' frame='true' align='right' caption='The bacterial biosynthetic thiolase: 1DM3, resolution 2.00 Å.'/>

[[Image:Fig4.png |left|thumb|Figure 3: Formation of the thioester enolate. A base is required for this proton abstraction. In thiolase this is Cys378. The oxyanion hole is formed by the Wat82-Asn316 diad and His348.]]
[[Image:Fig5.png |left| thumb|Figure 4: Formation of the tetrahedral intermediate thioester oxygen atom. The oxyanion hole is formed by two main chain NH-groups, from residues Cys89 and Gly380.]]
His348 has a
dual role, as it also activates Cys89 for nucleophilic attack.
A further key element of the active site geometry is <scene name='User:Satyan_Sharma/Sandbox_1/Oxyanion_hole_2/2'>oxyanion hole 2</scene>, which stabilizes the tetrahedral intermediate thioester oxyanion. This oxyanion hole is formed when the C2-atom of acetyl-CoA
reacts with the carbonyl-atom of the acetyl-cysteine thioester (Figure 4). The hydrogen bond donors of oxyanion hole 2 are N (Cys89) and N (Gly380).

The importance of the Asn316-Wat82 diad and His348 for the
oxyanion hole 1 stabilization of the transition state in the Claisen
condensation reaction has been confirmed by recent studies by
Merilainen ''et. al.''<ref name="merilainen" />

These key elements of the thiolase reaction mechanism have been
captured in the 1DM3 crystal structure,<ref>PMID:10764581</ref> which is a complex of
acetyl-CoA complexed with the acetylated enzyme (Figure 5). This
crystal form is grown at pH5. At this pH the catalytic base, Cys378,
is predicted to be always protonated, independent of the ligand bound
in the active site. Therefore it is unable to abstract the proton from
the methyl group of acetyl-CoA, and therefore the reaction
intermediate as shown in Figure 5 has been captured.
[[Image:Fig6.png | thumb|Figure 5. The captured intermediate (1DM3). In the active enzyme Cys378 is predicted to abstract a proton from the acetyl-CoA methyl group and in this way the Claisen condensation reaction is initiated. In this 1DM3-complex the pH is around 5 and therefore the intermediate has been trapped, as Cys378 will be predominantly protonated.]]

There are several structures known of the ''Zoogloea ramigera'' thiolase
complexed with the intermediates of the reaction cycle.<ref name="kursula" /> It is
interesting to point out that no gross structural changes of loops or
side chains have been detected. For example, the only structural
change with the apo structure (1DLU) is the small movement of the
Cys89 side chain towards the His348 side chain, by which it is assumed
that Cys89 is deprotonated and therefore activated.

== Diseases ==
The best characterized thiolase deficiencies concern mutations in the
T2 thiolase gene. T2 is important in the synthesis of ketone bodies
(in the mitochondria of the liver) as well as for the breakdown of
these ketone bodies, for example in the mitochondria of the brain and
heart. The T2 deficiency affects the breakdown of these ketone bodies
as is detected by accumulation of the oxidized ketone bodies in the
blood stream, causing ketoacidosis. This suggests that some other
thiolase is able to replace the synthetic, mitochondrial T2 thiolase
function, but not the degradation function. The T2 deficiencies have
been studied much by Dr. Fukao (Gifu University, Gifu, Japan).<ref name="Fukao" />

== Evolution ==

Thiolase turns out to be the founding member of a large superfamily of
enzymes, referred to as the thiolase superfamily.<ref>PMID:16356722</ref>
Key members of this superfamily are the KAS-enzymes. The KAS-enzyme is the first
enzyme in the fatty acid synthesis pathway. It has the same fold as
thiolase, and also the nucleophilic cysteine is conserved. Also
conserved in the KAS enzymes are the two residues which built oxyanion
hole 1 in thiolase. It has been noted that in the superfamily the
Asn316 and His348 either occur as a NH-pair (like in thiolase), or as
an HH-pair, or as an HN-pair, the NN-pair is never observed. Each of
the superfamily members also have the fully conserved cysteine
corresponding to Cys89. Therefore the members of the superfamily can
be divided into three categories, being CNH, CHH, and CHN.<ref>PMID:18824113</ref> In
the bacterial thiolase, extensive mutagenesis followed up by
structural enzymology<ref name="merilainen" /> has confirmed that the CNH-triad of
''Zoogloea ramigera'' is indeed important for the function of oxyanion
hole 1.

== References ==
<references/>

User:Satyan Sharma/Sandbox 1

2010-05-21T15:34:49Z

Satyan Sharma:

User:Satyan Sharma/Sandbox 1

2010-05-19T05:19:51Z

Satyan Sharma:

'''3D structure of the bacterial ''Zoogloea ramigera'' biosynthetic thiolase'''

== Introduction ==

Thiolase is best known as a key enzyme in the fatty acid degradation
pathway, also known as the β-oxidation pathway. However, in the human
metabolism at least six thiolases (Table 1) have been
characterized. Each of them important in different pathways, and in
different cell organelles. Some thiolases are dimers, some thiolases are
tetramers, being dimers of dimers. Thiolases are involved, either in
degradative pathways (E.C. 2.3.1.16), or in biosynthetic pathways
(E.C. 2.3.1.9). This division is somewhat arbitrary as all thiolases
are sequence related to each other and catalyse the reaction in both directions.

[[Image:Fig1.png|700px]]

The above scheme shows the degradative reaction catalysed by thiolase. In this the acetoacetyl-CoA is degraded with CoA as cosubstrate, by which two molecules of acetyl-CoA are formed. Each thiolase catalyzes this reaction in both directions, but the
equilibrium is far much favoring the degradative direction
(Keq is 105, favoring the degradation). The
reaction is catalyzed by the enzyme as a two step reaction. In the first step a fully conserved
active site cysteine, Cys89 is acetylated, liberating acetyl-CoA. In
the second step the acetyl-moiety is transferred to CoA.

The best studied thiolase is the bacterial ''Zoogloea ramigera'' thiolase,
which is a biosynthetic thiolase and which catalyzes the formation of
acetoacetyl-CoA from two molecules of acetyl-CoA. The enzymological
properties of ''Zoogloea ramigera'' thiolase have been extensively
characterized by C.T Walsh and coworkers<ref>PMID:1353760</ref> and more recently its
structural enzymological properties have been characterized also.<ref
name="kursula">PMID:12501183</ref><ref
name="merilainen">PMID:19842716</ref> The residue numbering on this page refers to the ''Zoogloea ramigera''
thiolase sequence, and the structural information concerns the 1DM3 structure.

[[Image:Reaction cycle.png|right|thumb|300px|Figure 1. The catalytic cycle of thiolase. The synthetic direction is the clockwise direction. In this direction first Cys89 is acetylated by acetyl-CoA. In the second step this acetyl group is transferred to acetyl-CoA, the Claisen condensation.]]

{| class="wikitable" style="text-align:center
|+ Table 1. The six identified human thiolases.
! width="125"|Intracellular localization
! width="125"|Mode of assembly
!width="125"|Function
!width="325"|Comment: thiolase protein code (gene sequence code)

|-
| cytosol
| α4 || synthesis || CT (THIC)
|-
| mitochondria
| α4 || synthesis and degradation || T2 (THIL) Many point mutation variants related to metabolic disorders have been characterized.<ref name="Fukao">PMID:20156697</ref>

|-
|
| α4 || degradation || T1 (THIM)
|-
|
| α4β4 || degradation || trifunctional enzyme, TFE (ECHB)
|-
| peroxisome
| α2 || degradation || A/B (THIK)
|-
|
| α2 || degradation || SCP2 (NLTP)
|}

== The reaction mechanism ==

[[Image:Fig3 tetramer.png|left|thumb|300px|Figure 2: the tetramer (1DM3)]]

The complete catalytic cycle of the ''Zoogloea ramigera'' thiolase is
shown in Figure 1. This thiolase is a tetramer, indeed a dimer of
dimers, as shown in Figure 2. The active site is deeply
buried. It is constructed by <scene name='User:Satyan_Sharma/Sandbox_1/Acsite_1dm3/2'>four loops</scene>, which provide
the catalytic nucleophile (Cys89), the base (Cys378), as well as
Asn316 and His348. Asn316 and His348 are key residues of the active
site geometry: a Asn316-Wat82 dyad, together with His348 make <scene name='User:Satyan_Sharma/Sandbox_1/Oxyanionhole_1/2'>oxyanion hole 1</scene>, stabilizing the CoA-thioester enolate intermediate. The hydrogen bond donors of oxyanion hole 1 are Wat82 and NE2 (His348). This enolate is formed from acetyl-CoA after proton
abstraction by the catalytic base, Cys378 (Figure 3).
<applet load='1dm3' size='300' frame='true' align='right' caption='The bacterial biosynthetic thiolase: 1DM3, resolution 2.00 Å.'/>

[[Image:Fig4.png |left|thumb|Figure 3: Formation of the thioester enolate. A base is required for this proton abstraction. In thiolase this is Cys378. The oxyanion hole is formed by the Wat82-Asn316 diad and His348.]]
[[Image:Fig5.png |left| thumb|Figure 4: Formation of the tetrahedral intermediate thioester oxygen atom. The oxyanion hole is formed by two main chain NH-groups, from residues Cys89 and Gly380.]]
His348 has a
dual role, as it also activates Cys89 for nucleophilic attack.
A further key element of the active site geometry is <scene name='User:Satyan_Sharma/Sandbox_1/Oxyanion_hole_2/2'>oxyanion hole 2</scene>, which stabilizes the tetrahedral intermediate thioester oxyanion. This oxyanion hole is formed when the C2-atom of acetyl-CoA
reacts with the carbonyl-atom of the acetyl-cysteine thioester (Figure 4). The hydrogen bond donors of oxyanion hole 2 are N (Cys89) and N (Gly380).

The importance of the Asn316-Wat82 diad and His348 for the
oxyanion hole 1 stabilization of the transition state in the Claisen
condensation reaction has been confirmed by recent studies by
Merilainen ''et. al.''<ref name="merilainen" />

These key elements of the thiolase reaction mechanism have been
captured in the 1DM3 crystal structure,<ref>PMID:10764581</ref> which is a complex of
acetyl-CoA complexed with the acetylated enzyme (Figure 5). This
crystal form is grown at pH5. At this pH the catalytic base, Cys378,
is predicted to be always protonated, independent of the ligand bound
in the active site. Therefore it is unable to abstract the proton from
the methyl group of acetyl-CoA, and therefore the reaction
intermediate as shown in Figure 5 has been captured.
[[Image:Fig6.png | thumb|Figure 5. The captured intermediate (1DM3). In the active enzyme Cys378 is predicted to abstract a proton from the acetyl-CoA methyl group and in this way the Claisen condensation reaction is initiated. In this 1DM3-complex the pH is around 5 and therefore the intermediate has been trapped, as Cys378 will be predominantly protonated.]]

There are several structures known of the ''Zoogloea ramigera'' thiolase
complexed with the intermediates of the reaction cycle.<ref name="kursula" /> It is
interesting to point out that no gross structural changes of loops or
side chains have been detected. For example, the only structural
change with the apo structure (1DLU) is the small movement of the
Cys89 side chain towards the His348 side chain, by which it is assumed
that Cys89 is deprotonated and therefore activated.

== Diseases ==
The best characterized thiolase deficiencies concern mutations in the
T2 thiolase gene. T2 is important in the synthesis of ketone bodies
(in the mitochondria of the liver) as well as for the breakdown of
these ketone bodies, for example in the mitochondria of the brain and
heart. The T2 deficiency affects the breakdown of these ketone bodies
as is detected by accumulation of the oxidized ketone bodies in the
blood stream, causing ketoacidosis. This suggests that some other
thiolase is able to replace the synthetic, mitochondrial T2 thiolase
function, but not the degradation function. The T2 deficiencies have
been studied much by Dr. Fukao (Gifu University, Gifu, Japan).<ref name="Fukao" />

== Evolution ==

Thiolase turns out to be the founding member of a large superfamily of
enzymes, referred to as the thiolase superfamily.<ref>PMID:16356722</ref>
Key members of this superfamily are the KAS-enzymes. The KAS-enzyme is the first
enzyme in the fatty acid synthesis pathway. It has the same fold as
thiolase, and also the nucleophilic cysteine is conserved. Also
conserved in the KAS enzymes are the two residues which built oxyanion
hole 1 in thiolase. It has been noted that in the superfamily the
Asn316 and His348 either occur as a NH-pair (like in thiolase), or as
an HH-pair, or as an HN-pair, the NN-pair is never observed. Each of
the superfamily members also have the fully conserved cysteine
corresponding to Cys89. Therefore the members of the superfamily can
be divided into three categories, being CNH, CHH, and CHN.<ref>PMID:18824113</ref> In
the bacterial thiolase, extensive mutagenesis followed up by
structural enzymology<ref name="merilainen" /> has confirmed that the CNH-triad of
''Zoogloea ramigera'' is indeed important for the function of oxyanion
hole 1.

== References ==
<references/>

User:Satyan Sharma/Sandbox 1

2010-05-19T05:17:36Z

Satyan Sharma:

'''The 1DM3-structure of the bacterial ''Zoogloea ramigera'' biosynthetic thiolase'''

== Introduction ==

Thiolase is best known as a key enzyme in the fatty acid degradation
pathway, also known as the β-oxidation pathway. However, in the human
metabolism at least six thiolases (Table 1) have been
characterized. Each of them important in different pathways, and in
different cell organelles. Some thiolases are dimers, some thiolases are
tetramers, being dimers of dimers. Thiolases are involved, either in
degradative pathways (E.C. 2.3.1.16), or in biosynthetic pathways
(E.C. 2.3.1.9). This division is somewhat arbitrary as all thiolases
are sequence related to each other and catalyse the reaction in both directions.

[[Image:Fig1.png|700px]]

The above scheme shows the degradative reaction catalysed by thiolase. In this the acetoacetyl-CoA is degraded with CoA as cosubstrate, by which two molecules of acetyl-CoA are formed. Each thiolase catalyzes this reaction in both directions, but the
equilibrium is far much favoring the degradative direction
(Keq is 105, favoring the degradation). The
reaction is catalyzed by the enzyme as a two step reaction. In the first step a fully conserved
active site cysteine, Cys89 is acetylated, liberating acetyl-CoA. In
the second step the acetyl-moiety is transferred to CoA.

The best studied thiolase is the bacterial ''Zoogloea ramigera'' thiolase,
which is a biosynthetic thiolase and which catalyzes the formation of
acetoacetyl-CoA from two molecules of acetyl-CoA. The enzymological
properties of ''Zoogloea ramigera'' thiolase have been extensively
characterized by C.T Walsh and coworkers<ref>PMID:1353760</ref> and more recently its
structural enzymological properties have been characterized also.<ref
name="kursula">PMID:12501183</ref><ref
name="merilainen">PMID:19842716</ref> The residue numbering on this page refers to the ''Zoogloea ramigera''
thiolase sequence, and the structural information concerns the 1DM3 structure.

[[Image:Reaction cycle.png|right|thumb|300px|Figure 1. The catalytic cycle of thiolase. The synthetic direction is the clockwise direction. In this direction first Cys89 is acetylated by acetyl-CoA. In the second step this acetyl group is transferred to acetyl-CoA, the Claisen condensation.]]

{| class="wikitable" style="text-align:center
|+ Table 1. The six identified human thiolases.
! width="125"|Intracellular localization
! width="125"|Mode of assembly
!width="125"|Function
!width="325"|Comment: thiolase protein code (gene sequence code)

|-
| cytosol
| α4 || synthesis || CT (THIC)
|-
| mitochondria
| α4 || synthesis and degradation || T2 (THIL) Many point mutation variants related to metabolic disorders have been characterized.<ref name="Fukao">PMID:20156697</ref>

|-
|
| α4 || degradation || T1 (THIM)
|-
|
| α4β4 || degradation || trifunctional enzyme, TFE (ECHB)
|-
| peroxisome
| α2 || degradation || A/B (THIK)
|-
|
| α2 || degradation || SCP2 (NLTP)
|}

== The reaction mechanism ==

[[Image:Fig3 tetramer.png|left|thumb|300px|Figure 2: the tetramer (1DM3)]]

The complete catalytic cycle of the ''Zoogloea ramigera'' thiolase is
shown in Figure 1. This thiolase is a tetramer, indeed a dimer of
dimers, as shown in Figure 2. The active site is deeply
buried. It is constructed by <scene name='User:Satyan_Sharma/Sandbox_1/Acsite_1dm3/2'>four loops</scene>, which provide
the catalytic nucleophile (Cys89), the base (Cys378), as well as
Asn316 and His348. Asn316 and His348 are key residues of the active
site geometry: a Asn316-Wat82 dyad, together with His348 make <scene name='User:Satyan_Sharma/Sandbox_1/Oxyanionhole_1/2'>oxyanion hole 1</scene>, stabilizing the CoA-thioester enolate intermediate. The hydrogen bond donors of oxyanion hole 1 are Wat82 and NE2 (His348). This enolate is formed from acetyl-CoA after proton
abstraction by the catalytic base, Cys378 (Figure 3).
<applet load='1dm3' size='300' frame='true' align='right' caption='The bacterial biosynthetic thiolase: 1DM3, resolution 2.00 Å.'/>

[[Image:Fig4.png |left|thumb|Figure 3: Formation of the thioester enolate. A base is required for this proton abstraction. In thiolase this is Cys378. The oxyanion hole is formed by the Wat82-Asn316 diad and His348.]]
[[Image:Fig5.png |left| thumb|Figure 4: Formation of the tetrahedral intermediate thioester oxygen atom. The oxyanion hole is formed by two main chain NH-groups, from residues Cys89 and Gly380.]]
His348 has a
dual role, as it also activates Cys89 for nucleophilic attack.
A further key element of the active site geometry is <scene name='User:Satyan_Sharma/Sandbox_1/Oxyanion_hole_2/2'>oxyanion hole 2</scene>, which stabilizes the tetrahedral intermediate thioester oxyanion. This oxyanion hole is formed when the C2-atom of acetyl-CoA
reacts with the carbonyl-atom of the acetyl-cysteine thioester (Figure 4). The hydrogen bond donors of oxyanion hole 2 are N (Cys89) and N (Gly380).

The importance of the Asn316-Wat82 diad and His348 for the
oxyanion hole 1 stabilization of the transition state in the Claisen
condensation reaction has been confirmed by recent studies by
Merilainen ''et. al.''<ref name="merilainen" />

These key elements of the thiolase reaction mechanism have been
captured in the 1DM3 crystal structure,<ref>PMID:10764581</ref> which is a complex of
acetyl-CoA complexed with the acetylated enzyme (Figure 5). This
crystal form is grown at pH5. At this pH the catalytic base, Cys378,
is predicted to be always protonated, independent of the ligand bound
in the active site. Therefore it is unable to abstract the proton from
the methyl group of acetyl-CoA, and therefore the reaction
intermediate as shown in Figure 5 has been captured.
[[Image:Fig6.png | thumb|Figure 5. The captured intermediate (1DM3). In the active enzyme Cys378 is predicted to abstract a proton from the acetyl-CoA methyl group and in this way the Claisen condensation reaction is initiated. In this 1DM3-complex the pH is around 5 and therefore the intermediate has been trapped, as Cys378 will be predominantly protonated.]]

There are several structures known of the ''Zoogloea ramigera'' thiolase
complexed with the intermediates of the reaction cycle.<ref name="kursula" /> It is
interesting to point out that no gross structural changes of loops or
side chains have been detected. For example, the only structural
change with the apo structure (1DLU) is the small movement of the
Cys89 side chain towards the His348 side chain, by which it is assumed
that Cys89 is deprotonated and therefore activated.

== Diseases ==
The best characterized thiolase deficiencies concern mutations in the
T2 thiolase gene. T2 is important in the synthesis of ketone bodies
(in the mitochondria of the liver) as well as for the breakdown of
these ketone bodies, for example in the mitochondria of the brain and
heart. The T2 deficiency affects the breakdown of these ketone bodies
as is detected by accumulation of the oxidized ketone bodies in the
blood stream, causing ketoacidosis. This suggests that some other
thiolase is able to replace the synthetic, mitochondrial T2 thiolase
function, but not the degradation function. The T2 deficiencies have
been studied much by Dr. Fukao (Gifu University, Gifu, Japan).<ref name="Fukao" />

== Evolution ==

Thiolase turns out to be the founding member of a large superfamily of
enzymes, referred to as the thiolase superfamily.<ref>PMID:16356722</ref>
Key members of this superfamily are the KAS-enzymes. The KAS-enzyme is the first
enzyme in the fatty acid synthesis pathway. It has the same fold as
thiolase, and also the nucleophilic cysteine is conserved. Also
conserved in the KAS enzymes are the two residues which built oxyanion
hole 1 in thiolase. It has been noted that in the superfamily the
Asn316 and His348 either occur as a NH-pair (like in thiolase), or as
an HH-pair, or as an HN-pair, the NN-pair is never observed. Each of
the superfamily members also have the fully conserved cysteine
corresponding to Cys89. Therefore the members of the superfamily can
be divided into three categories, being CNH, CHH, and CHN.<ref>PMID:18824113</ref> In
the bacterial thiolase, extensive mutagenesis followed up by
structural enzymology<ref name="merilainen" /> has confirmed that the CNH-triad of
''Zoogloea ramigera'' is indeed important for the function of oxyanion
hole 1.

== References ==
<references/>

User:Satyan Sharma/Sandbox 1

2010-05-19T05:08:10Z

Satyan Sharma:

== Introduction ==

Thiolase is best known as a key enzyme in the fatty acid degradation
pathway, also known as the β-oxidation pathway. However, in the human
metabolism at least six thiolases (Table 1) have been
characterized. Each of them important in different pathways, and in
different cell organelles. Some thiolases are dimers, some thiolases are
tetramers, being dimers of dimers. Thiolases are involved, either in
degradative pathways (E.C. 2.3.1.16), or in biosynthetic pathways
(E.C. 2.3.1.9). This division is somewhat arbitrary as all thiolases
are sequence related to each other and catalyse the reaction in both directions.

[[Image:Fig1.png|700px]]

The above scheme shows the degradative reaction catalysed by thiolase. In this the acetoacetyl-CoA is degraded with CoA as cosubstrate, by which two molecules of acetyl-CoA are formed. Each thiolase catalyzes this reaction in both directions, but the
equilibrium is far much favoring the degradative direction
(Keq is 105, favoring the degradation). The
reaction is catalyzed by the enzyme as a two step reaction. In the first step a fully conserved
active site cysteine, Cys89 is acetylated, liberating acetyl-CoA. In
the second step the acetyl-moiety is transferred to CoA.

The best studied thiolase is the bacterial ''Zoogloea ramigera'' thiolase,
which is a biosynthetic thiolase and which catalyzes the formation of
acetoacetyl-CoA from two molecules of acetyl-CoA. The enzymological
properties of ''Zoogloea ramigera'' thiolase have been extensively
characterized by C.T Walsh and coworkers<ref>PMID:1353760</ref> and more recently its
structural enzymological properties have been characterized also.<ref
name="kursula">PMID:12501183</ref><ref
name="merilainen">PMID:19842716</ref> The residue numbering on this page refers to the ''Zoogloea ramigera''
thiolase sequence, and the structural information concerns the 1DM3 structure.

[[Image:Reaction cycle.png|right|thumb|300px|Figure 1. The catalytic cycle of thiolase. The synthetic direction is the clockwise direction. In this direction first Cys89 is acetylated by acetyl-CoA. In the second step this acetyl group is transferred to acetyl-CoA, the Claisen condensation.]]

{| class="wikitable" style="text-align:center
|+ Table 1. The six identified human thiolases.
! width="125"|Intracellular localization
! width="125"|Mode of assembly
!width="125"|Function
!width="325"|Comment: thiolase protein code (gene sequence code)

|-
| cytosol
| α4 || synthesis || CT (THIC)
|-
| mitochondria
| α4 || synthesis and degradation || T2 (THIL) Many point mutation variants related to metabolic disorders have been characterized.<ref name="Fukao">PMID:20156697</ref>

|-
|
| α4 || degradation || T1 (THIM)
|-
|
| α4β4 || degradation || trifunctional enzyme, TFE (ECHB)
|-
| peroxisome
| α2 || degradation || A/B (THIK)
|-
|
| α2 || degradation || SCP2 (NLTP)
|}

== The reaction mechanism ==

[[Image:Fig3 tetramer.png|left|thumb|300px|Figure 2: the tetramer (1DM3)]]

The complete catalytic cycle of the ''Zoogloea ramigera'' thiolase is
shown in Figure 1. This thiolase is a tetramer, indeed a dimer of
dimers, as shown in Figure 2. The active site is deeply
buried. It is constructed by <scene name='User:Satyan_Sharma/Sandbox_1/Acsite_1dm3/2'>four loops</scene>, which provide
the catalytic nucleophile (Cys89), the base (Cys378), as well as
Asn316 and His348. Asn316 and His348 are key residues of the active
site geometry: a Asn316-Wat82 dyad, together with His348 make <scene name='User:Satyan_Sharma/Sandbox_1/Oxyanionhole_1/2'>oxyanion hole 1</scene>, stabilizing the CoA-thioester enolate intermediate. The hydrogen bond donors of oxyanion hole 1 are Wat82 and NE2 (His348). This enolate is formed from acetyl-CoA after proton
abstraction by the catalytic base, Cys378 (Figure 3).
<applet load='1dm3' size='300' frame='true' align='right' caption='The bacterial biosynthetic thiolase: 1DM3, resolution 2.00 Å.'/>

[[Image:Fig4.png |left|thumb|Figure 3: Formation of the thioester enolate. A base is required for this proton abstraction. In thiolase this is Cys378. The oxyanion hole is formed by the Wat82-Asn316 diad and His348.]]
[[Image:Fig5.png |left| thumb|Figure 4: Formation of the tetrahedral intermediate thioester oxygen atom. The oxyanion hole is formed by two main chain NH-groups, from residues Cys89 and Gly380.]]
His348 has a
dual role, as it also activates Cys89 for nucleophilic attack.
A further key element of the active site geometry is <scene name='User:Satyan_Sharma/Sandbox_1/Oxyanion_hole_2/2'>oxyanion hole 2</scene>, which stabilizes the tetrahedral intermediate thioester oxyanion. This oxyanion hole is formed when the C2-atom of acetyl-CoA
reacts with the carbonyl-atom of the acetyl-cysteine thioester (Figure 4). The hydrogen bond donors of oxyanion hole 2 are N (Cys89) and N (Gly380).

The importance of the Asn316-Wat82 diad and His348 for the
oxyanion hole 1 stabilization of the transition state in the Claisen
condensation reaction has been confirmed by recent studies by
Merilainen ''et. al.''<ref name="merilainen" />

These key elements of the thiolase reaction mechanism have been
captured in the 1DM3 crystal structure,<ref>PMID:10764581</ref> which is a complex of
acetyl-CoA complexed with the acetylated enzyme (Figure 5). This
crystal form is grown at pH5. At this pH the catalytic base, Cys378,
is predicted to be always protonated, independent of the ligand bound
in the active site. Therefore it is unable to abstract the proton from
the methyl group of acetyl-CoA, and therefore the reaction
intermediate as shown in Figure 5 has been captured.
[[Image:Fig6.png | thumb|Figure 5. The captured intermediate (1DM3). In the active enzyme Cys378 is predicted to abstract a proton from the acetyl-CoA methyl group and in this way the Claisen condensation reaction is initiated. In this 1DM3-complex the pH is around 5 and therefore the intermediate has been trapped, as Cys378 will be predominantly protonated.]]

There are several structures known of the ''Zoogloea ramigera'' thiolase
complexed with the intermediates of the reaction cycle.<ref name="kursula" /> It is
interesting to point out that no gross structural changes of loops or
side chains have been detected. For example, the only structural
change with the apo structure (1DLU) is the small movement of the
Cys89 side chain towards the His348 side chain, by which it is assumed
that Cys89 is deprotonated and therefore activated.

== Diseases ==
The best characterized thiolase deficiencies concern mutations in the
T2 thiolase gene. T2 is important in the synthesis of ketone bodies
(in the mitochondria of the liver) as well as for the breakdown of
these ketone bodies, for example in the mitochondria of the brain and
heart. The T2 deficiency affects the breakdown of these ketone bodies
as is detected by accumulation of the oxidized ketone bodies in the
blood stream, causing ketoacidosis. This suggests that some other
thiolase is able to replace the synthetic, mitochondrial T2 thiolase
function, but not the degradation function. The T2 deficiencies have
been studied much by Dr. Fukao (Gifu University, Gifu, Japan).<ref name="Fukao" />

== Evolution ==

Thiolase turns out to be the founding member of a large superfamily of
enzymes, referred to as the thiolase superfamily.<ref>PMID:16356722</ref>
Key members of this superfamily are the KAS-enzymes. The KAS-enzyme is the first
enzyme in the fatty acid synthesis pathway. It has the same fold as
thiolase, and also the nucleophilic cysteine is conserved. Also
conserved in the KAS enzymes are the two residues which built oxyanion
hole 1 in thiolase. It has been noted that in the superfamily the
Asn316 and His348 either occur as a NH-pair (like in thiolase), or as
an HH-pair, or as an HN-pair, the NN-pair is never observed. Each of
the superfamily members also have the fully conserved cysteine
corresponding to Cys89. Therefore the members of the superfamily can
be divided into three categories, being CNH, CHH, and CHN.<ref>PMID:18824113</ref> In
the bacterial thiolase, extensive mutagenesis followed up by
structural enzymology<ref name="merilainen" /> has confirmed that the CNH-triad of
''Zoogloea ramigera'' is indeed important for the function of oxyanion
hole 1.

== References ==
<references/>

User:Satyan Sharma/Sandbox 1

2010-05-18T09:10:05Z

Satyan Sharma:

{| style="background:yellow; color:black"
! UNDER CONSTRUCTION
|}

== Introduction ==

Thiolase is best known as a key enzyme in the fatty acid degradation
pathway, also known as the β-oxidation pathway. However, in the human
metabolism at least six thiolases (Table 1) have been
characterized. Each of them important in different pathways, and in
different cell organelles. Some thiolases are dimers, some thiolases are
tetramers, being dimers of dimers. Thiolases are involved, either in
degradative pathways (E.C. 2.3.1.16), or in biosynthetic pathways
(E.C. 2.3.1.9). This division is somewhat arbitrary as all thiolases
are sequence related to each other and catalyse the reaction in both directions.

[[Image:Fig1.png|700px]]

The above scheme shows the degradative reaction catalysed by thiolase. In this the acetoacetyl-CoA is degraded with CoA as cosubstrate, by which two molecules of acetyl-CoA are formed. Each thiolase catalyzes this reaction in both directions, but the
equilibrium is far much favoring the degradative direction
(Keq is 105, favoring the degradation). The
reaction is catalyzed by the enzyme as a two step reaction. In the first step a fully conserved
active site cysteine, Cys89 is acetylated, liberating acetyl-CoA. In
the second step the acetyl-moiety is transferred to CoA.

The best studied thiolase is the bacterial ''Zoogloea ramigera'' thiolase,
which is a biosynthetic thiolase and which catalyzes the formation of
acetoacetyl-CoA from two molecules of acetyl-CoA. The enzymological
properties of ''Zoogloea ramigera'' thiolase have been extensively
characterized by C.T Walsh and coworkers<ref>PMID:1353760</ref> and more recently its
structural enzymological properties have been characterized also.<ref
name="kursula">PMID:12501183</ref><ref
name="merilainen">PMID:19842716</ref> The residue numbering on this page refers to the ''Zoogloea ramigera''
thiolase sequence, and the structural information concerns the 1DM3 structure.

[[Image:Reaction cycle.png|right|thumb|300px|Figure 1. The catalytic cycle of thiolase. The synthetic direction is the clockwise direction. In this direction first Cys89 is acetylated by acetyl-CoA. In the second step this acetyl group is transferred to acetyl-CoA, the Claisen condensation.]]

{| class="wikitable" style="text-align:center
|+ Table 1. The six identified human thiolases.
! width="125"|Intracellular localization
! width="125"|Mode of assembly
!width="125"|Function
!width="325"|Comment: thiolase protein code (gene sequence code)

|-
| cytosol
| α4 || synthesis || CT (THIC)
|-
| mitochondria
| α4 || synthesis and degradation || T2 (THIL) Many point mutation variants related to metabolic disorders have been characterized.<ref name="Fukao">PMID:20156697</ref>

|-
|
| α4 || degradation || T1 (THIM)
|-
|
| α4β4 || degradation || trifunctional enzyme, TFE (ECHB)
|-
| peroxisome
| α2 || degradation || A/B (THIK)
|-
|
| α2 || degradation || SCP2 (NLTP)
|}

== The reaction mechanism ==

[[Image:Fig3 tetramer.png|left|thumb|300px|Figure 2: the tetramer (1DM3)]]

The complete catalytic cycle of the ''Zoogloea ramigera'' thiolase is
shown in Figure 1. This thiolase is a tetramer, indeed a dimer of
dimers, as shown in Figure 2. The active site is deeply
buried. It is constructed by <scene name='User:Satyan_Sharma/Sandbox_1/Acsite_1dm3/2'>four loops</scene>, which provide
the catalytic nucleophile (Cys89), the base (Cys378), as well as
Asn316 and His348. Asn316 and His348 are key residues of the active
site geometry: a Asn316-Wat82 dyad, together with His348 make <scene name='User:Satyan_Sharma/Sandbox_1/Oxyanionhole_1/2'>oxyanion hole 1</scene>, stabilizing the CoA-thioester enolate intermediate. The hydrogen bond donors of oxyanion hole 1 are Wat82 and NE2 (His348). This enolate is formed from acetyl-CoA after proton
abstraction by the catalytic base, Cys378 (Figure 3).
<applet load='1dm3' size='300' frame='true' align='right' caption='The bacterial biosynthetic thiolase: 1DM3, resolution 2.00 Å.'/>

[[Image:Fig4.png |left|thumb|Figure 3: Formation of the thioester enolate. A base is required for this proton abstraction. In thiolase this is Cys378. The oxyanion hole is formed by the Wat82-Asn316 diad and His348.]]
[[Image:Fig5.png |left| thumb|Figure 4: Formation of the tetrahedral intermediate thioester oxygen atom. The oxyanion hole is formed by two main chain NH-groups, from residues Cys89 and Gly380.]]
His348 has a
dual role, as it also activates Cys89 for nucleophilic attack.
A further key element of the active site geometry is <scene name='User:Satyan_Sharma/Sandbox_1/Oxyanion_hole_2/2'>oxyanion hole 2</scene>, which stabilizes the tetrahedral intermediate thioester oxyanion. This oxyanion hole is formed when the C2-atom of acetyl-CoA
reacts with the carbonyl-atom of the acetyl-cysteine thioester (Figure 4). The hydrogen bond donors of oxyanion hole 2 are N (Cys89) and N (Gly380).

The importance of the Asn316-Wat82 diad and His348 for the
oxyanion hole 1 stabilization of the transition state in the Claisen
condensation reaction has been confirmed by recent studies by
Merilainen ''et. al.''<ref name="merilainen" />

These key elements of the thiolase reaction mechanism have been
captured in the 1DM3 crystal structure,<ref>PMID:10764581</ref> which is a complex of
acetyl-CoA complexed with the acetylated enzyme (Figure 5). This
crystal form is grown at pH5. At this pH the catalytic base, Cys378,
is predicted to be always protonated, independent of the ligand bound
in the active site. Therefore it is unable to abstract the proton from
the methyl group of acetyl-CoA, and therefore the reaction
intermediate as shown in Figure 5 has been captured.
[[Image:Fig6.png | thumb|Figure 5. The captured intermediate (1DM3). In the active enzyme Cys378 is predicted to abstract a proton from the acetyl-CoA methyl group and in this way the Claisen condensation reaction is initiated. In this 1DM3-complex the pH is around 5 and therefore the intermediate has been trapped, as Cys378 will be predominantly protonated.]]

There are several structures known of the ''Zoogloea ramigera'' thiolase
complexed with the intermediates of the reaction cycle.<ref name="kursula" /> It is
interesting to point out that no gross structural changes of loops or
side chains have been detected. For example, the only structural
change with the apo structure (1DLU) is the small movement of the
Cys89 side chain towards the His348 side chain, by which it is assumed
that Cys89 is deprotonated and therefore activated.

== Diseases ==
The best characterized thiolase deficiencies concern mutations in the
T2 thiolase gene. T2 is important in the synthesis of ketone bodies
(in the mitochondria of the liver) as well as for the breakdown of
these ketone bodies, for example in the mitochondria of the brain and
heart. The T2 deficiency affects the breakdown of these ketone bodies
as is detected by accumulation of the oxidized ketone bodies in the
blood stream, causing ketoacidosis. This suggests that some other
thiolase is able to replace the synthetic, mitochondrial T2 thiolase
function, but not the degradation function. The T2 deficiencies have
been studied much by Dr. Fukao (Gifu University, Gifu, Japan).<ref name="Fukao" />

== Evolution ==

Thiolase turns out to be the founding member of a large superfamily of
enzymes, referred to as the thiolase superfamily.<ref>PMID:16356722</ref>
Key members of this superfamily are the KAS-enzymes. The KAS-enzyme is the first
enzyme in the fatty acid synthesis pathway. It has the same fold as
thiolase, and also the nucleophilic cysteine is conserved. Also
conserved in the KAS enzymes are the two residues which built oxyanion
hole 1 in thiolase. It has been noted that in the superfamily the
Asn316 and His348 either occur as a NH-pair (like in thiolase), or as
an HH-pair, or as an HN-pair, the NN-pair is never observed. Each of
the superfamily members also have the fully conserved cysteine
corresponding to Cys89. Therefore the members of the superfamily can
be divided into three categories, being CNH, CHH, and CHN.<ref>PMID:18824113</ref> In
the bacterial thiolase, extensive mutagenesis followed up by
structural enzymology<ref name="merilainen" /> has confirmed that the CNH-triad of
''Zoogloea ramigera'' is indeed important for the function of oxyanion
hole 1.

== References ==
<references/>

2010-04-08T05:43:23Z

Satyan Sharma:

{| style="background:yellow; color:black"
! UNDER CONSTRUCTION
|}
<applet load='1dm3' size='300' frame='true' align='right' caption='Insert caption here' />

== Introduction ==

Thiolase is best known as key enzyme in the β-oxidation of fatty acid degradation pathway. However, in the human metabolism at least six thiolases (Table 1) have been characterized. Each of them is important in different pathways, and in different organelles. Some of the thiolases are dimers, some thiolases are tetramers, being dimers of dimers. Thiolases are involved, either in degradative pathways (E.C.2.3.1.16), or in biosynthetic pathways (E.C.2.3.1.9). This division is somewhat arbitrary as all thiolases are aequence related to each other.

Figure 1 shows the thiolase reaction in the degradative direction. In the degradative reaction acetoacetyl-CoA is degraded with CoA as cosubstrate and by which two molecules of acetoacetyl-CoA are formed. Each thiolase catalyzes this reaction in both directions, but the equilibrium is far much favouring the degradative direction (Keq is 105, favoring the degradation). The reaction is catalysed by the enzyme as a two step process. In the first step a fully conserved active site cystiene, Cys89 is acetylated liberating acetyl-CoA. In the second step the acetyl-moiety is transferred to CoA (Figure 1).

{| class="wikitable" style="text-align:center
|+ Table 1. Mammalian thiolases: localization and function.
! width="325"|Thiolase
! width="250"|Cellular localization
!width="325"|Function
|-
| Mitochondrial acetoacetyl-CoA thiolase (T2)
| mitochondria || Ketogenesis (in liver), Ketolysis (in extrahepatic tissues)
|-
| Mitochondrial 3-ketoacyl-CoA thiolase (T1)
| mitochondrial matrix || β-oxidation, ketogenesis
|-
| TFE
| mitochondrial inner membrane || β-oxidation
|-
| Cytosolic thiolase
| cytosol || Cholesterol synthesis
|-
| 3-ketoacyl-CoA thiolase (A/B-thiolase)
| peroxisome || Peroxisomal β-oxidation
|-
|SCP2/thiolase
| perosisome || Peroxisomal β-oxidation of branched chain fatty acids, bile acid metabolism
|}

The best studied thiolase is the ''Zoogloea ramigera'' thiolase, which is a biosynthetic thiolase and catalyzes the formation of actoacetyl-CoA from two molecules of acetyl-CoA (Figure 2). The enzymological properties of ''Zoogloea ramigera'' thiolase have been extensively characterized by C.T. Wals and coworkers<ref>PMID:1353760</ref> and more recently its structural enzymologiacl properties have also been characterized.<ref>PMID:12501183</ref>

== The reaction mechanism ==

The complete catalytic cycle of the ''Zoogloea ramigera'' thiolase, is shown in Figure 3. This thiolase is a tetramer, indeed a dimer of dimers. The <scene name='User:Satyan_Sharma/Sandbox_1/Acsite/1'>active site</scene> is deeply buried. It is constructed by four loops, which provide the catalytic nucleophile (Cys89), the base (Cys378), as well as Asn316 and His348.Asn316 and His348 are key residues of the active site geometry: a Asn316-Wat82 diad, together with His348 makes oxyanion hole 1 (green), stabilizing the CoA-thioester enolate intermediate. This enolate is formed on proton abstraction by the catalytic base, Cys378, from acetyl-CoA (Figure 5). His348 has a dual role, as it also activates Cys89 for nucleophilic attack. Further key element of the active site geometry is oxyanion hole 2 (green), which stabilizes the tetrahedral intermediate thioester oxyanion, which is formed when the C2-atom of acetyl-CoA reacts with the carbonyl-atom of the acetyl-Cystiene thioester (Figure 6). The importance of the Asn316-Wat82 diad and His348 for the oxyanion hole 1 stabilization of the transition state in the Claisen condensation reaction has been confirmed by recent studies by Merilainen ''et. al.''.<ref>PMID:19842716</ref>

These key elements of the thiolase reaction mechanism have been capture in the 1DM3 crystal structure,<ref>PMID:10785370</ref> which is a complex of acetyl-CoA complexed with the acetylated enzyme (green). This crystal form is grown at pH 5. At this pH the catalytic base is predicted to be always protonated, independent of the ligand bound in the active site. Therefore it is unable to abstract the proton from the methyl group of acetyl-CoA, and therefore the reaction intermediate, as shown in Figure 7, has been captured.

There are several structures known ''Zoogloea ramigera'' thiolase complexed with the intermediates of the reaction cycle.<ref>PMID:12501183</ref> It is interesting to point out that no gross structural changes of loops or side chains have been detected. For example, the only gross structural change with the apo structure (1DLU) is the small movement of the Cys89 side chain towards the His348 side chain, by which it is assumed that Cys89 is deprotonated and therefore activated.

==Diseases==

The best characterized thiolase deficiencies concern mutations in the T2 thiolase. T2 is important in the sysnthesis of ketone bodies (in the liver, in mitochondria) as well as for the breakdown of these ketone bodies, for example in the mitochondria of the brain. The T2 deficiency affects the breakdown of these ketone bodies as is detected by the accumulation of the oxidized ketone bodies in the blood stream, causing ketoacidosis. This suggests that some other thiolase is able to replace synthetic, mitochondrial T2 thiolase fucntion, but not the degradation function in the brain. The T2 deficiencies have been studied much by Dr. Fukao (Gifu University, Gifu, Japan).<ref>PMID:20156697</ref>

==Evolution==

Thiolase turns out to be the founding member of a large superfamily of enzymes, referred to as the thiolase superfamily. Key member of this superfamily are the KAS-enzymes. The KAS-enzyme is the first enzyme in the fatty acid synthesis pathway. It has the same fold as thiolase, and also the nucleophilic cystiene is conserved. Also conserved in the KAS-enzymes are the two residues which built oxynaion hole 1 in thiolase. It has been noted that in the superfamily, the Asn316 and His348 either occur as a NH-pair (like in thiolase), or as a HH-pair or as a HN-pair. The NN-pair is not conserved. Therefore, the members of the superfamily can be divided into three categories, being CNH, CHH, and CHN.<ref>PMID: 18824113</ref> In the bacterial thiolase, extensive mutagenesis followed up by structural enzymology<ref>PMID:19842716</ref> has confirmed that the CNH-triad of ''Zoogloea ramigera'' is indeed important for the function of oxyanion hole 1.

==References==
<references/>

User:Satyan Sharma/Sandbox 1

2010-04-07T09:35:10Z

Satyan Sharma:

'''UNDER CONSTRUCTION'''
<applet load='1dm3' size='300' frame='true' align='right' caption='Insert caption here' />
== Introduction ==

Thiolase is best known as key enzyme in the β-oxidation of fatty acid degradation pathway. However, in the human metabolism at least six thiolases have been characterized. Each of them is important in different pathways, and in different organelles. Some of the thiolases are dimers, some thiolases are tetramers, being dimers of dimers. Thiolases are involved, either in degradative pathways (E.C.2.3.1.16), or in biosynthetic pathways (E.C.2.3.1.9). This division is somewhat arbitrary as all thiolases are aequence related to each other.

Figure 1 shows the thiolase reaction in the degradative direction. In the degradative reaction acetoacetyl-CoA is degraded with CoA as cosubstrate and by which two molecules of acetoacetyl-CoA are formed. Each thiolase catalyzes this reaction in both directions, but the equilibrium is far much favouring the degradative direction (Keq is 105, favoring the degradation). The reaction is catalysed by the enzyme as a two step process. In the first step a fully conserved active site cystiene, Cys89 is acetylated liberating acetyl-CoA. In the second step the acetyl-moiety is transferred to CoA (Figure 1).

The best studied thiolase is the ''Zoogloea ramigera'' thiolase, which is a biosynthetic thiolase and catalyzes the formation of actoacetyl-CoA from two molecules of acetyl-CoA (Figure 2). The enzymological properties of ''Zoogloea ramigera'' thiolase have been extensively characterized by C.T. Wals and coworkers<ref>PMID:1353760</ref> and more recently its structural enzymologiacl properties have also been characterized.<ref>PMID:12501183</ref>

== The reaction mechanism ==

The complete catalytic cycle of the ''Zoogloea ramigera'' thiolase, is shown in Figure 3. This thiolase is a tetramer, indeed a dimer of dimers. The <scene name='User:Satyan_Sharma/Sandbox_1/Acsite/1'>active site</scene> is deeply buried. It is constructed by four loops, which provide the catalytic nucleophile (Cys89), the base (Cys378), as well as Asn316 and His348.Asn316 and His348 are key residues of the active site geometry: a Asn316-Wat82 diad, together with His348 makes oxyanion hole 1 (green), stabilizing the CoA-thioester enolate intermediate. This enolate is formed on proton abstraction by the catalytic base, Cys378, from acetyl-CoA (Figure 5). His348 has a dual role, as it also activates Cys89 for nucleophilic attack. Further key element of the active site geometry is oxyanion hole 2 (green), which stabilizes the tetrahedral intermediate thioester oxyanion, which is formed when the C2-atom of acetyl-CoA reacts with the carbonyl-atom of the acetyl-Cystiene thioester (Figure 6). The importance of the Asn316-Wat82 diad and His348 for the oxyanion hole 1 stabilization of the transition state in the Claisen condensation reaction has been confirmed by recent studies by Merilainen ''et. al.''.<ref>PMID:19842716</ref>

These key elements of the thiolase reaction mechanism have been capture in the 1DM3 crystal structure,<ref>PMID:10785370</ref> which is a complex of acetyl-CoA complexed with the acetylated enzyme (green). This crystal form is grown at pH 5. At this pH the catalytic base is predicted to be always protonated, independent of the ligand bound in the active site. Therefore it is unable to abstract the proton from the methyl group of acetyl-CoA, and therefore the reaction intermediate, as shown in Figure 7, has been captured.

There are several structures known ''Zoogloea ramigera'' thiolase complexed with the intermediates of the reaction cycle.<ref>PMID:12501183</ref> It is interesting to point out that no gross structural changes of loops or side chains have been detected. For example, the only gross structural change with the apo structure (1DLU) is the small movement of the Cys89 side chain towards the His348 side chain, by which it is assumed that Cys89 is deprotonated and therefore activated.

==Diseases==

The best characterized thiolase deficiencies concern mutations in the T2 thiolase. T2 is important in the sysnthesis of ketone bodies (in the liver, in mitochondria) as well as for the breakdown of these ketone bodies, for example in the mitochondria of the brain. The T2 deficiency affects the breakdown of these ketone bodies as is detected by the accumulation of the oxidized ketone bodies in the blood stream, causing ketoacidosis. This suggests that some other thiolase is able to replace synthetic, mitochondrial T2 thiolase fucntion, but not the degradation function in the brain. The T2 deficiencies have been studied much by Dr. Fukao (Gifu University, Gifu, Japan).<ref>PMID:20156697</ref>

==Evolution==

Thiolase turns out to be the founding member of a large superfamily of enzymes, referred to as the thiolase superfamily. Key member of this superfamily are the KAS-enzymes. The KAS-enzyme is the first enzyme in the fatty acid synthesis pathway. It has the same fold as thiolase, and also the nucleophilic cystiene is conserved. Also conserved in the KAS-enzymes are the two residues which built oxynaion hole 1 in thiolase. It has been noted that in the superfamily, the Asn316 and His348 either occur as a NH-pair (like in thiolase), or as a HH-pair or as a HN-pair. The NN-pair is not conserved. Therefore, the members of the superfamily can be divided into three categories, being CNH, CHH, and CHN.<ref>PMID: 18824113</ref> In the bacterial thiolase, extensive mutagenesis followed up by structural enzymology<ref>PMID:19842716</ref> has confirmed that the CNH-triad of ''Zoogloea ramigera'' is indeed important for the function of oxyanion hole 1.

==References==
<references/>