Nitrile hydratase - Revision history

Michal Harel at 07:57, 18 July 2023

Michal Harel — Tue, 18 Jul 2023 07:57:33 GMT

←Older revision		Revision as of 07:57, 18 July 2023
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	**[[1ire]], [[1ugq]], [[4ob0]], [[4ob3]] – PtNH α,β subunits – ''Pseudonocardia thermophila''<br />		**[[1ire]], [[1ugq]], [[4ob0]], [[4ob3]] – PtNH α,β subunits – ''Pseudonocardia thermophila''<br />
-	**[[1ugr]], [[1ugs]], [[3vyh]] – PtNH α (mutant),β subunits <br />	+	**[[1ugr]], [[1ugs]], [[3vyh]], [[7sjz]], [[7w8l]] – PtNH α (mutant),β subunits <br />
		+	**[[7qom]] – PtNH α,β (mutant) subunits <br />
		+	**[[7qom]], [[7qou]], [[7qoy]] – PtNH α (mutant),β (mutant) subunits <br />
	**[[1v29]] – NH α,β subunits – ''Bacillus smithii''<br />		**[[1v29]] – NH α,β subunits – ''Bacillus smithii''<br />
	**[[2dpp]] – NH α,β subunits – ''Bacillus'' <br />		**[[2dpp]] – NH α,β subunits – ''Bacillus'' <br />
	**[[3qxe]], [[3qyg]], [[3qyh]], [[3qz5]], [[3qz9]] – NH α,β subunits – ''Pseudomonas putida''<br />		**[[3qxe]], [[3qyg]], [[3qyh]], [[3qz5]], [[3qz9]] – NH α,β subunits – ''Pseudomonas putida''<br />
-	**[[~~3hht~~]] – NH α (mutant),β ~~(mutant)~~ subunits – ''Geobacillus palilidus''<br />	+	**[[7qop]] – GpNH α (mutant),β subunits – ''Geobacillus palilidus''<br />
		+	**[[3hht]], [[7z0v]] – GpNH α (mutant),β (mutant) subunits <br />
	**[[1ugp]] – PtNH α,β subunits + butyrate<br />		**[[1ugp]] – PtNH α,β subunits + butyrate<br />
	**[[4ob1]], [[4ob2]] – PtNH α,β subunits + butane boronic acid<br />		**[[4ob1]], [[4ob2]] – PtNH α,β subunits + butane boronic acid<br />

Michal Harel at 09:46, 23 November 2021

Michal Harel — Tue, 23 Nov 2021 09:46:37 GMT

←Older revision		Revision as of 09:46, 23 November 2021
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	**[[4fm4]], [[4zgd]] – CtNH α,β subunits – ''Comamonas testosteroni''<br />		**[[4fm4]], [[4zgd]] – CtNH α,β subunits – ''Comamonas testosteroni''<br />
	**[[4zge]], [[4zgj]] – CtNH α (mutant),β subunits <br />		**[[4zge]], [[4zgj]] – CtNH α (mutant),β subunits <br />
		+	**[[6lk7]] – NH α subunit – ''Pseudomonas''<br />

	*Fe-containing nitrile hydratase complex		*Fe-containing nitrile hydratase complex

Michal Harel at 11:25, 10 November 2019

Michal Harel — Sun, 10 Nov 2019 11:25:23 GMT

←Older revision		Revision as of 11:25, 10 November 2019
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	**[[1v29]] – NH α,β subunits – ''Bacillus smithii''<br />		**[[1v29]] – NH α,β subunits – ''Bacillus smithii''<br />
	**[[2dpp]] – NH α,β subunits – ''Bacillus'' <br />		**[[2dpp]] – NH α,β subunits – ''Bacillus'' <br />
-	**[[3qxe]], [[3qyg]], [[3qz5]], [[3qz9]] – NH α,β subunits – ''Pseudomonas putida''<br />	+	**[[3qxe]], [[3qyg]], [[3qyh]], [[3qz5]], [[3qz9]] – NH α,β subunits – ''Pseudomonas putida''<br />
	**[[3hht]] – NH α (mutant),β (mutant) subunits – ''Geobacillus palilidus''<br />		**[[3hht]] – NH α (mutant),β (mutant) subunits – ''Geobacillus palilidus''<br />
	**[[1ugp]] – PtNH α,β subunits + butyrate<br />		**[[1ugp]] – PtNH α,β subunits + butyrate<br />

Michal Harel at 09:26, 24 August 2017

Michal Harel — Thu, 24 Aug 2017 09:26:03 GMT

←Older revision		Revision as of 09:26, 24 August 2017
Line 67:		Line 67:
	**[[3a8g]] – ReNH α (mutant),β subunits + trimethyl acetonitrile + NO <br />		**[[3a8g]] – ReNH α (mutant),β subunits + trimethyl acetonitrile + NO <br />
	**[[3a8h]] – ReNH α (mutant),β subunits + trimethyl acetonitrile <br />		**[[3a8h]] – ReNH α (mutant),β subunits + trimethyl acetonitrile <br />
-	**[[3a8m]], [[3wvd, [[3wve]], [[3x20]], [[3x24]], [[3x25]], [[3x26]], [[3x28]] – ReNH α,β (mutant) subunits + trimethyl acetonitrile <br />	+	**[[3a8m]], [[3wvd]], [[3wve]], [[3x20]], [[3x24]], [[3x25]], [[3x26]], [[3x28]] – ReNH α,β (mutant) subunits + trimethyl acetonitrile <br />

	*'''Co-containing nitrile hydratase'''		*'''Co-containing nitrile hydratase'''

Michal Harel at 09:25, 24 August 2017

Michal Harel — Thu, 24 Aug 2017 09:25:13 GMT

←Older revision		Revision as of 09:25, 24 August 2017
Line 53:		Line 53:
	**[[1ahj]], [[2cyz]], [[2cz0]], [[2cz1]] – ReNH α,β subunits – ''Rhodococcus erythropolis''<br />		**[[1ahj]], [[2cyz]], [[2cz0]], [[2cz1]] – ReNH α,β subunits – ''Rhodococcus erythropolis''<br />
	**[[2zcf]], [[3a8l]] – ReNH α (mutant),β subunits <br />		**[[2zcf]], [[3a8l]] – ReNH α (mutant),β subunits <br />
-	**[[4fm4]] – NH α,β subunits – ''Comamonas testosteroni''<br />	+	**[[4fm4]], [[4zgd]] – CtNH α,β subunits – ''Comamonas testosteroni''<br />
		+	**[[4zge]], [[4zgj]] – CtNH α (mutant),β subunits <br />

	*Fe-containing nitrile hydratase complex		*Fe-containing nitrile hydratase complex
Line 66:		Line 67:
	**[[3a8g]] – ReNH α (mutant),β subunits + trimethyl acetonitrile + NO <br />		**[[3a8g]] – ReNH α (mutant),β subunits + trimethyl acetonitrile + NO <br />
	**[[3a8h]] – ReNH α (mutant),β subunits + trimethyl acetonitrile <br />		**[[3a8h]] – ReNH α (mutant),β subunits + trimethyl acetonitrile <br />
-	**[[3a8m]] – ReNH α,β (mutant) subunits + trimethyl acetonitrile <br />	+	**[[3a8m]], [[3wvd, [[3wve]], [[3x20]], [[3x24]], [[3x25]], [[3x26]], [[3x28]] – ReNH α,β (mutant) subunits + trimethyl acetonitrile <br />

	*'''Co-containing nitrile hydratase'''		*'''Co-containing nitrile hydratase'''

Michal Harel at 09:49, 15 May 2016

Michal Harel — Sun, 15 May 2016 09:49:15 GMT

←Older revision		Revision as of 09:49, 15 May 2016
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	'''Nitrile hydratase''' (NH) catalyze the hydration of nitriles to amides. NH contains Fe or Co ion in its active site. NH consists of two subunits: α and β and exists as dimer or tetramer with one metal ion bound per dimer.		'''Nitrile hydratase''' (NH) catalyze the hydration of nitriles to amides. NH contains Fe or Co ion in its active site. NH consists of two subunits: α and β and exists as dimer or tetramer with one metal ion bound per dimer.
-
-	== Disease ==

	== Relevance ==		== Relevance ==

Alexander Berchansky at 12:07, 9 August 2015

Alexander Berchansky — Sun, 09 Aug 2015 12:07:04 GMT

←Older revision		Revision as of 12:07, 9 August 2015
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	These data implicate the specificity of substrate binding for a definite member of Branch 10 of the Nit-nitrilase superfamily. The present solution of the substrate-free bacterial protein structure of SA0302 gives the basis for substrate binding studies with the potential substrates or inhibitors.		These data implicate the specificity of substrate binding for a definite member of Branch 10 of the Nit-nitrilase superfamily. The present solution of the substrate-free bacterial protein structure of SA0302 gives the basis for substrate binding studies with the potential substrates or inhibitors.

-	=== Analyzing the Catalytic Role of Active Site Residues in the Fe-Type Nitrile Hydratase from ''Comamonas testosteroni'' Ni1 ~~===~~	+	=== Analyzing the Catalytic Role of Active Site Residues in the Fe-Type Nitrile Hydratase from ''Comamonas testosteroni'' Ni1 <ref>doi 10.1007/s00775-015-1273-3</ref>===
-	~~<big>Salette Martinez, Rui Wu, Karoline Krzywda, Veronika Opalka, Hei Chan, Dali Liu, and~~	+
-	~~Richard C. Holz</big>~~ <ref>doi 10.1007/s00775-015-1273-3</ref>	+
-	~~<hr/>~~	+

	<scene name='70/703434/Cv/3'>Active site of wild-type Fe-type nitrile hydratase</scene> from ''Comamonas testosteroni'' Ni1 (CtNHase, PDB ID: [[4fm4]]) in ball-and-stick form is shown. A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the CtNHase, <scene name='70/703434/Cv/8'>were mutated</scene>. <font color='darkmagenta'><b>The wild-type is in dark magenta</b></font>, <span style="color:lime;background-color:black;font-weight:bold;">αH80A/αH81A mutant is in green</span>, <span style="color:orange;background-color:black;font-weight:bold;">αH80W/αH81W is in orange</span>, and the <span style="color:deepskyblue;background-color:black;font-weight:bold;">αR157A mutant is in deep sky blue</span>. <scene name='70/703434/Cv/9'>Click here to see animation of this scene</scene>. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k<sub>cat</sub> = 10 ± 2 s−1) accounts for less than 1 % of the wild-type activity (k<sub>cat</sub> = 1100 ± 30 s<sup>-1</sup>) while the K<sub>m</sub> value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k<sub>cat</sub> values of 220 ± 40 and 77 ± 13 s<sup>-1</sup>, respectively, and K<sub>m</sub> values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k<sub>cat</sub> value of 132 ± 3 s<sup>-1</sup> and a K<sub>m</sub> value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the <scene name='70/703434/Cv/11'>αH80A/αH81A</scene>, <scene name='70/703434/Cv/12'>αH80W/αH81W</scene>, and <scene name='70/703434/Cv/13'>αR157A</scene> mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, <scene name='70/703434/Cv/14'>hydrogen-bonding interactions crucial for the catalytic function of the αCys104-SOH ligand are disrupted</scene>. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.		<scene name='70/703434/Cv/3'>Active site of wild-type Fe-type nitrile hydratase</scene> from ''Comamonas testosteroni'' Ni1 (CtNHase, PDB ID: [[4fm4]]) in ball-and-stick form is shown. A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the CtNHase, <scene name='70/703434/Cv/8'>were mutated</scene>. <font color='darkmagenta'><b>The wild-type is in dark magenta</b></font>, <span style="color:lime;background-color:black;font-weight:bold;">αH80A/αH81A mutant is in green</span>, <span style="color:orange;background-color:black;font-weight:bold;">αH80W/αH81W is in orange</span>, and the <span style="color:deepskyblue;background-color:black;font-weight:bold;">αR157A mutant is in deep sky blue</span>. <scene name='70/703434/Cv/9'>Click here to see animation of this scene</scene>. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k<sub>cat</sub> = 10 ± 2 s−1) accounts for less than 1 % of the wild-type activity (k<sub>cat</sub> = 1100 ± 30 s<sup>-1</sup>) while the K<sub>m</sub> value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k<sub>cat</sub> values of 220 ± 40 and 77 ± 13 s<sup>-1</sup>, respectively, and K<sub>m</sub> values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k<sub>cat</sub> value of 132 ± 3 s<sup>-1</sup> and a K<sub>m</sub> value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the <scene name='70/703434/Cv/11'>αH80A/αH81A</scene>, <scene name='70/703434/Cv/12'>αH80W/αH81W</scene>, and <scene name='70/703434/Cv/13'>αR157A</scene> mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, <scene name='70/703434/Cv/14'>hydrogen-bonding interactions crucial for the catalytic function of the αCys104-SOH ligand are disrupted</scene>. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

Alexander Berchansky at 12:05, 9 August 2015

Alexander Berchansky — Sun, 09 Aug 2015 12:05:52 GMT

←Older revision		Revision as of 12:05, 9 August 2015
Line 39:		Line 39:

	These data implicate the specificity of substrate binding for a definite member of Branch 10 of the Nit-nitrilase superfamily. The present solution of the substrate-free bacterial protein structure of SA0302 gives the basis for substrate binding studies with the potential substrates or inhibitors.		These data implicate the specificity of substrate binding for a definite member of Branch 10 of the Nit-nitrilase superfamily. The present solution of the substrate-free bacterial protein structure of SA0302 gives the basis for substrate binding studies with the potential substrates or inhibitors.
		+
		+	=== Analyzing the Catalytic Role of Active Site Residues in the Fe-Type Nitrile Hydratase from ''Comamonas testosteroni'' Ni1 ===
		+	<big>Salette Martinez, Rui Wu, Karoline Krzywda, Veronika Opalka, Hei Chan, Dali Liu, and
		+	Richard C. Holz</big> <ref>doi 10.1007/s00775-015-1273-3</ref>
		+	<hr/>
		+
		+	<scene name='70/703434/Cv/3'>Active site of wild-type Fe-type nitrile hydratase</scene> from ''Comamonas testosteroni'' Ni1 (CtNHase, PDB ID: [[4fm4]]) in ball-and-stick form is shown. A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the CtNHase, <scene name='70/703434/Cv/8'>were mutated</scene>. <font color='darkmagenta'><b>The wild-type is in dark magenta</b></font>, <span style="color:lime;background-color:black;font-weight:bold;">αH80A/αH81A mutant is in green</span>, <span style="color:orange;background-color:black;font-weight:bold;">αH80W/αH81W is in orange</span>, and the <span style="color:deepskyblue;background-color:black;font-weight:bold;">αR157A mutant is in deep sky blue</span>. <scene name='70/703434/Cv/9'>Click here to see animation of this scene</scene>. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k<sub>cat</sub> = 10 ± 2 s−1) accounts for less than 1 % of the wild-type activity (k<sub>cat</sub> = 1100 ± 30 s<sup>-1</sup>) while the K<sub>m</sub> value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k<sub>cat</sub> values of 220 ± 40 and 77 ± 13 s<sup>-1</sup>, respectively, and K<sub>m</sub> values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k<sub>cat</sub> value of 132 ± 3 s<sup>-1</sup> and a K<sub>m</sub> value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the <scene name='70/703434/Cv/11'>αH80A/αH81A</scene>, <scene name='70/703434/Cv/12'>αH80W/αH81W</scene>, and <scene name='70/703434/Cv/13'>αR157A</scene> mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, <scene name='70/703434/Cv/14'>hydrogen-bonding interactions crucial for the catalytic function of the αCys104-SOH ligand are disrupted</scene>. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.
		+	<scene name='70/703434/Cv/4'>Superposition of the αβ heterodimer</scene>, the regions with pronounced difference between the wild-type and the mutant enzymes are indicated. <scene name='70/703434/Cv/7'>Click here to see animation of this scene</scene>.
	</StructureSection>		</StructureSection>

Alexander Berchansky at 12:01, 10 May 2015

Alexander Berchansky — Sun, 10 May 2015 12:01:37 GMT

←Older revision		Revision as of 12:01, 10 May 2015
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	== Structural highlights ==		== Structural highlights ==

-	=== Crystal structure of the CN-hydrolase SA0302 from the pathogenic bacterium ''Staphylococcus aureus'' belonging to the Nit and NitFhit Branch of the nitrilase superfamily ~~===~~	+	=== Crystal structure of the CN-hydrolase SA0302 from the pathogenic bacterium ''Staphylococcus aureus'' belonging to the Nit and NitFhit Branch of the nitrilase superfamily <ref>doi 10.1080/07391102.2012.719111</ref>===
-	~~<big>Roni D. Gordon, Wei Qiu, Vladimir Romanov, Kim Lam, Maria Soloveychik, Diana Benetteraj, Kevin P. Battaile, Yuri N. Chirgadze, Emil F. Pai, and Nickolay Y. Chirgadze</big>~~ <ref>doi 10.1080/07391102.2012.719111</ref>	+
-	~~<hr/>~~	+
-	~~<b>Molecular Tour</b><br>~~	+
	The nitrilase, superfamily of protein enzyme, has in total over 180 known members. It includes a variety of thiol amidase enzymes involved in biosynthesis in plants, animals, fungi and prokaryotes <ref name="Pace">PMID:11380987</ref>.		The nitrilase, superfamily of protein enzyme, has in total over 180 known members. It includes a variety of thiol amidase enzymes involved in biosynthesis in plants, animals, fungi and prokaryotes <ref name="Pace">PMID:11380987</ref>.

Alexander Berchansky at 12:00, 10 May 2015

Alexander Berchansky — Sun, 10 May 2015 12:00:33 GMT

←Older revision		Revision as of 12:00, 10 May 2015
Line 12:		Line 12:

	== Structural highlights ==		== Structural highlights ==
		+
		+	=== Crystal structure of the CN-hydrolase SA0302 from the pathogenic bacterium ''Staphylococcus aureus'' belonging to the Nit and NitFhit Branch of the nitrilase superfamily ===
		+	<big>Roni D. Gordon, Wei Qiu, Vladimir Romanov, Kim Lam, Maria Soloveychik, Diana Benetteraj, Kevin P. Battaile, Yuri N. Chirgadze, Emil F. Pai, and Nickolay Y. Chirgadze</big> <ref>doi 10.1080/07391102.2012.719111</ref>
		+	<hr/>
		+	<b>Molecular Tour</b><br>
		+	The nitrilase, superfamily of protein enzyme, has in total over 180 known members. It includes a variety of thiol amidase enzymes involved in biosynthesis in plants, animals, fungi and prokaryotes <ref name="Pace">PMID:11380987</ref>.
		+
		+	All members of this superfamily have conserved the active site residues Glu-Lys-Cys, believed to form a catalytic triad. The consensus sequences flanking the catalytic residues supply the conserved motifs distinctive for all of the branches <ref name="Pace"/>. The superfamily can be classified into 13 branches, nine of which have known or predicted specificity for nitrilase, amidase, and CN-hydrolase reactions. Although the entire family has been considered “nitrilase-related”, only members of Branch 1 have demonstrated nitrilase activity. The remaining branches include enzymes with amidase or amide-condensation activity including aliphatic amidase, amino-terminal amidase, biotinidase, β-ureidopropionase, carbamylase, prokaryotic and eukaryotic NAD-synthetase, and apo-lipoprotein N-acyltransferase <ref name="Brenner">PMID:12504683</ref>. Analysis of the sequences and structures of CN-hydrolases with known three-dimensional structures shows that SA0302 definitely is a member of Branch 10 (Nit and NitFhit) of the nitrilase superfamily. Enzyme activities and substrate specificities of members of this branch are not yet characterized, in contrast to those of the members of Branches 1 to 9.
		+
		+	The monomer consists of <scene name='Journal:JBSD:19/Cv/4'>two similar sub-domains related by a pseudo two-fold rotational symmetry</scene> <span style="color:salmon;background-color:black;font-weight:bold;">N-subdomain is colored in salmon</span> and <font color='magenta'><b>C-subdomain is in magenta</b></font>. The <scene name='Journal:JBSD:19/Cv/5'>active site</scene> is formed by the residues from both the N- and C-terminal sub-domains (catalytic residues are labeled). The protein contains 261 amino acids and consists of a four-layer <scene name='Journal:JBSD:19/Cv/6'>αββα sandwich</scene> (color-coding: {{Template:ColorKey_Helix}}, {{Template:ColorKey_Strand}}, {{Template:ColorKey_Turn}}), consistent with the expected CN-hydrolase fold. The <scene name='Journal:JBSD:19/Cv/7'>dimer</scene>, which was found in crystal and solution media, contains two accessible to the solvent active sites. <span style="color:lime;background-color:black;font-weight:bold;">First monomer is colored in green</span>, <font color='darkmagenta'><b>second monomer is in darkmagenta</b></font>, <font color='red'><b>positions of catalytic triad residues are in red</b></font>, and the <span style="color:orange;background-color:black;font-weight:bold;">conserved α6-β10 hairpin is shown in orange</span>.
		+
		+	The sequence alignments of SA0302 with four known nitrilase structures are presented in the image below.
		+	[[Image:Fig2jbsd19.png\|left\|400px\|thumb\|]]
		+	{{Clear}}
		+	They clearly show the <scene name='Journal:JBSD:19/Cv/8'>conserved catalytic triad Glu41-Lys110-Cys146</scene> which believed to describe the enzyme active site. These catalytic residues are located within <scene name='Journal:JBSD:19/Cv/9'>three semi-conserved regions</scene> corresponding to their relative locations in the SA0302 protein sequence: region 1 – flanking E41; region 2 – following K110; and region 3 – flanking C146, which also has a protein signature consistent with the 21-residue “nucleophile elbow” motif <ref name="Kumaran">PMID:12833551</ref>. These flanking catalytic sequences of SA0302 are in good accordance with consensus sequences of CN-hydrolase of Branch 10 (Nit and NitFhit) of the nitrilase superfamily <ref name="Pace"/>. On the basis of semi-conserved regions flanking the catalytic triad, we have assigned ten other uncharacterized protein sequences to Branch 10 of the nitrilase superfamily (see image below).
		+	[[Image:Fig3.png\|left\|400px\|thumb\|]]
		+
		+	{{Clear}}
		+	<scene name='Journal:JBSD:19/Cv/11'>General view of the active site</scene> with the catalytic triad. <scene name='Journal:JBSD:19/Cv/10'>Detailed analysis</scene> shows that active site includes five participants: catalytic triad Glu41-Lys110-Cys146, water molecule W272, and additional residue Glu119.
		+
		+	In spite of growing interest about the details of the enzymatic mechanism of the members of Branch 10, at present little is known about the specificity of possible substrates or inhibitors. This is a very challenging biochemical problem that is still far from being resolved. At the moment, we can cite one important related reference <ref name="Barglow">PMID:19053248</ref>. In this paper, two murine nitrilases, including Nit1 and Nit2, were identified as targets for a dipeptide-chloroacetamide activity-based probe. The gel analysis of binding of Nit with these probes shows definite selectivity of labeling inside the Nit subfamily. Experimental data of positive labeling are as follows:
		+	Nit1: '''Leu-Tyr''', '''Leu-His''', '''Leu-Leu''', Asp-Leu, Glu-Leu, Tyr-Leu, '''D-Leu-Asp''', Leu-D-Asp, D-Leu-D-Asp, D-Leu-D-Asp
		+
		+	Nit2: '''Leu-Tyr''', '''Leu-His''', '''Leu-Leu''', Leu-Arg, Leu-Glu, Leu-Asp, '''D-Leu-Asp''',
		+
		+	where common dipeptide α-CA probes are in bold.
		+
		+	These data implicate the specificity of substrate binding for a definite member of Branch 10 of the Nit-nitrilase superfamily. The present solution of the substrate-free bacterial protein structure of SA0302 gives the basis for substrate binding studies with the potential substrates or inhibitors.
	</StructureSection>		</StructureSection>