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Structure of R-specific alcohol dehydrogenase (mutant G37D) from Lactobacillus brevis in complex with phenylethanol and NAD

OverviewOverview

The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is an NADP-dependent, homotetrameric member of the extended enzyme family of short-chain dehydrogenases/reductases (SDR) with a high biotechnological application potential. Its preferred in vitro substrates are prochiral ketones like acetophenone with almost invariably a small methyl group as one substituent and a bulky (often aromatic) moiety as the other. On the basis of an atomic-resolution structure of wild-type RADH in complex with NADP and acetophenone, we designed the mutant RADH-G37D, which should possess an improved cosubstrate specificity profile for biotechnological purposes, namely, a preference for NAD rather than NADP. Comparative kinetic measurements with wild-type and mutant RADH showed that this aim was achieved. To characterize the successful mutant structurally, we determined several, partly atomic-resolution, crystal structures of RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends on an interaction between the adenosine ribose moiety of NAD and the inserted aspartate side-chain. A structural comparison between RADH-G37D as apo-enzyme and as a part of a ternary complex revealed significant rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the active site. This plasticity contributes to generate a small hydrophobic pocket for the methyl group typical for RADH substrates, and a hydrophobic coat for the second, more variable and often aromatic, substituent. Around Ser141 we even found alternative conformations in the backbone. A structural adaptability in this region, which we describe here for the first time for an SDR enzyme, is probably functionally important, because it concerns Ser142, a member of the highly conserved catalytic tetrad typical for SDR enzymes. Moreover, it affects an extended proton relay system that has been identified recently as a critical element for the catalytic mechanism in SDR enzymes.

About this StructureAbout this Structure

1ZK1 is a Single protein structure of sequence from Lactobacillus brevis with , and as ligands. Active as Alcohol dehydrogenase (NADP(+)), with EC number 1.1.1.2 Full crystallographic information is available from OCA.

ReferenceReference

Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity., Schlieben NH, Niefind K, Muller J, Riebel B, Hummel W, Schomburg D, J Mol Biol. 2005 Jun 17;349(4):801-13. PMID:15896805

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-[[Image:1zk1.gif|left|200px]]<br /><applet load="1zk1" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1zk1.gif|left|200px]]<br /><applet load="1zk1" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1zk1, resolution 1.78&Aring;" />
 '''Structure of R-specific alcohol dehydrogenase (mutant G37D) from Lactobacillus brevis in complex with phenylethanol and NAD'''<br />
 ==Overview==
-The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is, an NADP-dependent, homotetrameric member of the extended enzyme family of, short-chain dehydrogenases/reductases (SDR) with a high biotechnological, application potential. Its preferred in vitro substrates are prochiral, ketones like acetophenone with almost invariably a small methyl group as, one substituent and a bulky (often aromatic) moiety as the other. On the, basis of an atomic-resolution structure of wild-type RADH in complex with, NADP and acetophenone, we designed the mutant RADH-G37D, which should, possess an improved cosubstrate specificity profile for biotechnological, purposes, namely, a preference for NAD rather than NADP. Comparative, kinetic measurements with wild-type and mutant RADH showed that this aim, was achieved. To characterize the successful mutant structurally, we, determined several, partly atomic-resolution, crystal structures of, RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH, and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends, on an interaction between the adenosine ribose moiety of NAD and the, inserted aspartate side-chain. A structural comparison between RADH-G37D, as apo-enzyme and as a part of a ternary complex revealed significant, rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the, active site. This plasticity contributes to generate a small hydrophobic, pocket for the methyl group typical for RADH substrates, and a hydrophobic, coat for the second, more variable and often aromatic, substituent. Around, Ser141 we even found alternative conformations in the backbone. A, structural adaptability in this region, which we describe here for the, first time for an SDR enzyme, is probably functionally important, because, it concerns Ser142, a member of the highly conserved catalytic tetrad, typical for SDR enzymes. Moreover, it affects an extended proton relay, system that has been identified recently as a critical element for the, catalytic mechanism in SDR enzymes.
+The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is an NADP-dependent, homotetrameric member of the extended enzyme family of short-chain dehydrogenases/reductases (SDR) with a high biotechnological application potential. Its preferred in vitro substrates are prochiral ketones like acetophenone with almost invariably a small methyl group as one substituent and a bulky (often aromatic) moiety as the other. On the basis of an atomic-resolution structure of wild-type RADH in complex with NADP and acetophenone, we designed the mutant RADH-G37D, which should possess an improved cosubstrate specificity profile for biotechnological purposes, namely, a preference for NAD rather than NADP. Comparative kinetic measurements with wild-type and mutant RADH showed that this aim was achieved. To characterize the successful mutant structurally, we determined several, partly atomic-resolution, crystal structures of RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends on an interaction between the adenosine ribose moiety of NAD and the inserted aspartate side-chain. A structural comparison between RADH-G37D as apo-enzyme and as a part of a ternary complex revealed significant rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the active site. This plasticity contributes to generate a small hydrophobic pocket for the methyl group typical for RADH substrates, and a hydrophobic coat for the second, more variable and often aromatic, substituent. Around Ser141 we even found alternative conformations in the backbone. A structural adaptability in this region, which we describe here for the first time for an SDR enzyme, is probably functionally important, because it concerns Ser142, a member of the highly conserved catalytic tetrad typical for SDR enzymes. Moreover, it affects an extended proton relay system that has been identified recently as a critical element for the catalytic mechanism in SDR enzymes.
 ==About this Structure==
-ZK1 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Lactobacillus_brevis Lactobacillus brevis] with MG, NAD and AC0 as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alcohol_dehydrogenase_(NADP(+)) Alcohol dehydrogenase (NADP(+))], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.2 1.1.1.2] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1ZK1 OCA].
+ZK1 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Lactobacillus_brevis Lactobacillus brevis] with <scene name='pdbligand=MG:'>MG</scene>, <scene name='pdbligand=NAD:'>NAD</scene> and <scene name='pdbligand=AC0:'>AC0</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alcohol_dehydrogenase_(NADP(+)) Alcohol dehydrogenase (NADP(+))], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.2 1.1.1.2] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1ZK1 OCA].
 ==Reference==
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 [[Category: Niefind, K.]]
 [[Category: Riebel, B.]]
-[[Category: Schlieben, N.H.]]
+[[Category: Schlieben, N H.]]
 [[Category: Schomburg, D.]]
 [[Category: AC0]]
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 [[Category: short chain reductases/dehydrogenases]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Sun Nov 25 04:54:52 2007''
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