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Understanding oligomerization in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: An in silico approach and evidence for an active protein

Paper ID Volume ID Publish Year Pages File Format Full-Text
25260 43563 2007 9 PDF Available
Title
Understanding oligomerization in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: An in silico approach and evidence for an active protein
Abstract

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni belongs to the short chain dehydrogenase/reductase (SDR) protein superfamily and catalyzes the oxidoreduction of a variety of steroid substrates, including the steroid antibiotic fusidic acid. The enzyme also mediates the carbonyl reduction of non-steroidal aldehydes and ketones such as a novel insecticide. It is suggested that 3α-HSD/CR contributes to the bioremediation of natural and synthetic toxicants by C. testosteroni. Crystallization and structure analysis showed that 3α-HSD/CR is active as a dimer. Dimerization takes place via an interface axis which has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3α-HSD/CR by the presence of a predominantly α-helical subdomain which is missing in all other SDRs of known structure. For example, 3α/20β-HSD from Streptomyces hydrogenans exhibits two main subunit interfaces arranged about two non-crystallographic two-fold axes which are perpendicular to each other and referred to as P and Q. This mode of dimerization is, however, sterically impossible in 3α-HSD/CR because of a 28 amino acids insertion into the classical Rossmann-fold motif between strand βE and helix αF. This insertion is masking helices αE and αF, thus preventing the formation of a four helix bundle and enables the dimerization via a P-axis interface. This type of dimerization in SDRs has never been observed in a crystal structure so far.The aim of this study was to investigate whether the lack of this predominantly α-helical subdomain keeps 3α-HSD/CR to be an active enzyme and whether, by an in silico approach, the formation of a homotetramer or even a novel oligomerization mode can be expected. Redesign of this interface was performed on the basis of site directed mutagenesis and according to other SDR structures by an approach combining “in silico” and “wet chemistry”. Simulations of sterical and structural effects after different mutations, by applying a combination of homology modelling and molecular dynamic simulations, provided an effective tool for extensive mutagenesis studies and indicated the possibility of tetramer formation of truncated 3α-HSD/CR. In addition, despite lacking the extra loop domain, mutant 3α-HSD/CR was shown to be active towards a variety of standard substrates.

Keywords
SDR; 3α-HSD/CR; MD-simulation; Dimerization
First Page Preview
Understanding oligomerization in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: An in silico approach and evidence for an active protein
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 129, Issue 1, 30 March 2007, Pages 131–139
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering