Publications by authors named "René M de Jong"

19 Publications

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Macromolecular modeling and design in Rosetta: recent methods and frameworks.

Authors:
Julia Koehler Leman Brian D Weitzner Steven M Lewis Jared Adolf-Bryfogle Nawsad Alam Rebecca F Alford Melanie Aprahamian David Baker Kyle A Barlow Patrick Barth Benjamin Basanta Brian J Bender Kristin Blacklock Jaume Bonet Scott E Boyken Phil Bradley Chris Bystroff Patrick Conway Seth Cooper Bruno E Correia Brian Coventry Rhiju Das René M De Jong Frank DiMaio Lorna Dsilva Roland Dunbrack Alexander S Ford Brandon Frenz Darwin Y Fu Caleb Geniesse Lukasz Goldschmidt Ragul Gowthaman Jeffrey J Gray Dominik Gront Sharon Guffy Scott Horowitz Po-Ssu Huang Thomas Huber Tim M Jacobs Jeliazko R Jeliazkov David K Johnson Kalli Kappel John Karanicolas Hamed Khakzad Karen R Khar Sagar D Khare Firas Khatib Alisa Khramushin Indigo C King Robert Kleffner Brian Koepnick Tanja Kortemme Georg Kuenze Brian Kuhlman Daisuke Kuroda Jason W Labonte Jason K Lai Gideon Lapidoth Andrew Leaver-Fay Steffen Lindert Thomas Linsky Nir London Joseph H Lubin Sergey Lyskov Jack Maguire Lars Malmström Enrique Marcos Orly Marcu Nicholas A Marze Jens Meiler Rocco Moretti Vikram Khipple Mulligan Santrupti Nerli Christoffer Norn Shane Ó'Conchúir Noah Ollikainen Sergey Ovchinnikov Michael S Pacella Xingjie Pan Hahnbeom Park Ryan E Pavlovicz Manasi Pethe Brian G Pierce Kala Bharath Pilla Barak Raveh P Douglas Renfrew Shourya S Roy Burman Aliza Rubenstein Marion F Sauer Andreas Scheck William Schief Ora Schueler-Furman Yuval Sedan Alexander M Sevy Nikolaos G Sgourakis Lei Shi Justin B Siegel Daniel-Adriano Silva Shannon Smith Yifan Song Amelie Stein Maria Szegedy Frank D Teets Summer B Thyme Ray Yu-Ruei Wang Andrew Watkins Lior Zimmerman Richard Bonneau

Nat Methods 2020 07 1;17(7):665-680. Epub 2020 Jun 1.

Center for Computational Biology, Flatiron Institute, Simons Foundation, New York, NY, USA.

The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities. Here we review tools developed in the last 5 years, including over 80 methods. We discuss improvements to the score function, user interfaces and usability. Rosetta is available at http://www.rosettacommons.org.
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http://dx.doi.org/10.1038/s41592-020-0848-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603796PMC
July 2020

Rhodoxanthin synthase from honeysuckle; a membrane diiron enzyme catalyzes the multistep conversation of β-carotene to rhodoxanthin.

Sci Adv 2020 Apr 22;6(17):eaay9226. Epub 2020 Apr 22.

DSM Nutritional Products, 60 Westview St, Lexington, MA 02421, USA.

Rhodoxanthin is a vibrant red carotenoid found across the plant kingdom and in certain birds and fish. It is a member of the atypical retro class of carotenoids, which contain an additional double bond and a concerted shift of the conjugated double bonds relative to the more widely occurring carotenoid pigments, and whose biosynthetic origins have long remained elusive. Here, we identify LHRS ( hydroxylase rhodoxanthin synthase), a variant β-carotene hydroxylase (BCH)-type integral membrane diiron enzyme that mediates the conversion of β-carotene into rhodoxanthin. We identify residues that are critical to rhodoxanthin formation by LHRS. Substitution of only three residues converts a typical BCH into a multifunctional enzyme that mediates a multistep pathway from β-carotene to rhodoxanthin via a series of distinct oxidation steps in which the product of each step becomes the substrate for the next catalytic cycle. We propose a biosynthetic pathway from β-carotene to rhodoxanthin.
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http://dx.doi.org/10.1126/sciadv.aay9226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176425PMC
April 2020

Metal Dependence of the Xylose Isomerase from Piromyces sp. E2 Explored by Activity Profiling and Protein Crystallography.

Biochemistry 2017 11 2;56(45):5991-6005. Epub 2017 Nov 2.

Biochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , Nijenborgh 4, 9747 AG Groningen, The Netherlands.

Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn, which was established by measuring the activation constants (K) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity.
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http://dx.doi.org/10.1021/acs.biochem.7b00777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688467PMC
November 2017

CorNet: Assigning function to networks of co-evolving residues by automated literature mining.

PLoS One 2017 18;12(5):e0176427. Epub 2017 May 18.

Bio-Prodict, Nijmegen, The Netherlands.

CorNet is a web-based tool for the analysis of co-evolving residue positions in protein super-family sequence alignments. CorNet projects external information such as mutation data extracted from literature on interactively displayed groups of co-evolving residue positions to shed light on the functions associated with these groups and the residues in them. We used CorNet to analyse six enzyme super-families and found that groups of strongly co-evolving residues tend to consist of residues involved in a same function such as activity, specificity, co-factor binding, or enantioselectivity. This finding allows to assign a function to residues for which no data is available yet in the literature. A mutant library was designed to mutate residues observed in a group of co-evolving residues predicted to be involved in enantioselectivity, but for which no literature data is available yet. The resulting set of mutations indeed showed many instances of increased enantioselectivity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176427PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436653PMC
September 2017

Semi-rational engineering of cytochrome CYP153A from Marinobacter aquaeolei for improved ω-hydroxylation activity towards oleic acid.

Appl Microbiol Biotechnol 2016 Oct 27;100(20):8779-88. Epub 2016 May 27.

Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.

ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket. The in vitro-reconstituted activity of Variant II with purified Pdx and Pdr was 2.7-fold that of the template, while the whole cell transformation activity was 2.0-fold that of the template. A 96-well format-based screening scheme for CYP153A was also developed, which should be useful for engineering of other P450s with low activity. Kinetic analyses indicated that the activity improvement for CYP153A variants largely resulted from enhanced electron transfer. This further demonstrates the importance of the electron transfer between P450s and the non-native redox partners for the overall performance of hybrid P450 systems.
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http://dx.doi.org/10.1007/s00253-016-7634-1DOI Listing
October 2016

An engineered cryptic Hxt11 sugar transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae.

Biotechnol Biofuels 2015 2;8:176. Epub 2015 Nov 2.

Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, Nijenborgh 7, 9747 AG Groningen, The Netherlands.

Background: The yeast Saccharomyces cerevisiae is unable to ferment pentose sugars like d-xylose. Through the introduction of the respective metabolic pathway, S. cerevisiae is able to ferment xylose but first utilizes d-glucose before the d-xylose can be transported and metabolized. Low affinity d-xylose uptake occurs through the endogenous hexose (Hxt) transporters. For a more robust sugar fermentation, co-consumption of d-glucose and d-xylose is desired as d-xylose fermentation is in particular prone to inhibition by compounds present in pretreated lignocellulosic feedstocks.

Results: Evolutionary engineering of a d-xylose-fermenting S. cerevisiae strain lacking the major transporter HXT1-7 and GAL2 genes yielded a derivative that shows improved growth on xylose because of the expression of a normally cryptic HXT11 gene. Hxt11 also supported improved growth on d-xylose by the wild-type strain. Further selection for glucose-insensitive growth on d-xylose employing a quadruple hexokinase deletion yielded mutations at N366 of Hxt11 that reversed the transporter specificity for d-glucose into d-xylose while maintaining high d-xylose transport rates. The Hxt11 mutant enabled the efficient co-fermentation of xylose and glucose at industrially relevant sugar concentrations when expressed in a strain lacking the HXT1-7 and GAL2 genes.

Conclusions: Hxt11 is a cryptic sugar transporter of S. cerevisiae that previously has not been associated with effective d-xylose transport. Mutagenesis of Hxt11 yielded transporters that show a better affinity for d-xylose as compared to d-glucose while maintaining high transport rates. d-glucose and d-xylose co-consumption is due to a redistribution of the sugar transport flux while maintaining the total sugar conversion rate into ethanol. This method provides a single transporter solution for effective fermentation on lignocellulosic feedstocks.
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http://dx.doi.org/10.1186/s13068-015-0360-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4630928PMC
November 2015

Coupling Protein Side-Chain and Backbone Flexibility Improves the Re-design of Protein-Ligand Specificity.

PLoS Comput Biol 2015 23;11(9):e1004335. Epub 2015 Sep 23.

Graduate Program in Bioinformatics, University of California San Francisco, San Francisco, California, United States of America; California Institute for Quantitative Biosciences (QB3), University of California San Francisco, San Francisco, California, United States of America; Department of Bioengineering and Therapeutic Science, University of California San Francisco, San Francisco, California, United States of America.

Interactions between small molecules and proteins play critical roles in regulating and facilitating diverse biological functions, yet our ability to accurately re-engineer the specificity of these interactions using computational approaches has been limited. One main difficulty, in addition to inaccuracies in energy functions, is the exquisite sensitivity of protein-ligand interactions to subtle conformational changes, coupled with the computational problem of sampling the large conformational search space of degrees of freedom of ligands, amino acid side chains, and the protein backbone. Here, we describe two benchmarks for evaluating the accuracy of computational approaches for re-engineering protein-ligand interactions: (i) prediction of enzyme specificity altering mutations and (ii) prediction of sequence tolerance in ligand binding sites. After finding that current state-of-the-art "fixed backbone" design methods perform poorly on these tests, we develop a new "coupled moves" design method in the program Rosetta that couples changes to protein sequence with alterations in both protein side-chain and protein backbone conformations, and allows for changes in ligand rigid-body and torsion degrees of freedom. We show significantly increased accuracy in both predicting ligand specificity altering mutations and binding site sequences. These methodological improvements should be useful for many applications of protein-ligand design. The approach also provides insights into the role of subtle conformational adjustments that enable functional changes not only in engineering applications but also in natural protein evolution.
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http://dx.doi.org/10.1371/journal.pcbi.1004335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580623PMC
March 2016

Engineering of an endogenous hexose transporter into a specific D-xylose transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae.

Biotechnol Biofuels 2014 29;7(1):168. Epub 2014 Nov 29.

Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, Groningen, The Netherlands.

Background: Engineering of Saccharomyces cerevisiae for the simultaneous utilization of hexose and pentose sugars is vital for cost-efficient cellulosic bioethanol production. This yeast lacks specific pentose transporters and depends on endogenous hexose transporters for low affinity pentose uptake. Consequently, engineered xylose-fermenting yeast strains first utilize D-glucose before D-xylose can be transported and metabolized.

Results: We have used an evolutionary engineering approach that depends on a quadruple hexokinase deletion xylose-fermenting S. cerevisiae strain to select for growth on D-xylose in the presence of high D-glucose concentrations. This resulted in D-glucose-tolerant growth of the yeast of D-xylose. This could be attributed to mutations at N367 in the endogenous chimeric Hxt36 transporter, causing a defect in D-glucose transport while still allowing specific uptake of D-xylose. The Hxt36-N367A variant transports D-xylose with a high rate and improved affinity, enabling the efficient co-consumption of D-glucose and D-xylose.

Conclusions: Engineering of yeast endogenous hexose transporters provides an effective strategy to construct glucose-insensitive xylose transporters that are well integrated in the carbon metabolism regulatory network, and that can be used for efficient lignocellulosic bioethanol production.
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http://dx.doi.org/10.1186/s13068-014-0168-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263072PMC
December 2014

Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis).

FEBS Lett 2014 Mar 11;588(6):1001-7. Epub 2014 Feb 11.

Plant Research International, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. Electronic address:

(+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was detected at 144±10μg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases.
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http://dx.doi.org/10.1016/j.febslet.2014.01.061DOI Listing
March 2014

Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.

Plant Biotechnol J 2014 Feb 25;12(2):174-82. Epub 2013 Sep 25.

Plant Research International, Wageningen, the Netherlands; Platform Green Synthetic Biology, Delft University of Technology, Delft, the Netherlands.

Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential.
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http://dx.doi.org/10.1111/pbi.12124DOI Listing
February 2014

Saturation mutagenesis of Asn152 reveals a substrate selectivity switch in P99 cephalosporinase.

Protein Sci 2007 Dec;16(12):2636-46

Department of Biological Sciences, Columbia University, New York, New York 10027, USA.

In class C beta-lactamases, the strictly conserved Asn152 forms part of an extended active-site hydrogen-bonding network. To probe its role in catalysis, all 19 mutants of Enterobacter cloacae P99 cephalosporinase Asn152 were simultaneously constructed and screened in Escherichia coli for their in vivo activity. The screen identified the previously uncharacterized mutants Asn152Ser, Asn152Thr, and Asn152Gly, which possess significant activity and altered substrate selectivity. In vitro measurement of Michaelis-Menten kinetic constants revealed that the Asn152Ser mutation causes a selectivity switch for penicillin G versus cefoxitin. Asn152Thr showed a 63-fold increase in k (cat) for oxacillin, a slow substrate for wild-type cephalosporinase. The results contribute to a growing body of data showing that mutation of highly conserved residues in the active site can result in substrate selectivity changes. The library screening method presented here would be applicable to substrate selectivity determination in other readily screenable enzymes.
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http://dx.doi.org/10.1110/ps.073092407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2222824PMC
December 2007

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase. Structural basis for substrate specificity and inactivation by (R)-oxirane-2-carboxylate.

J Biol Chem 2007 Jan 22;282(4):2440-9. Epub 2006 Nov 22.

Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103'). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103' activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103' distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor.
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http://dx.doi.org/10.1074/jbc.M608134200DOI Listing
January 2007

The X-ray structure of the haloalcohol dehalogenase HheA from Arthrobacter sp. strain AD2: insight into enantioselectivity and halide binding in the haloalcohol dehalogenase family.

J Bacteriol 2006 Jun;188(11):4051-6

Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

Haloalcohol dehalogenases are bacterial enzymes that cleave the carbon-halogen bond in short aliphatic vicinal haloalcohols, like 1-chloro-2,3-propanediol, some of which are recalcitrant environmental pollutants. They use a conserved Ser-Tyr-Arg catalytic triad to deprotonate the haloalcohol oxygen, which attacks the halogen-bearing carbon atom, producing an epoxide and a halide ion. Here, we present the X-ray structure of the haloalcohol dehalogenase HheA(AD2) from Arthrobacter sp. strain AD2 at 2.0-A resolution. Comparison with the previously reported structure of the 34% identical enantioselective haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 shows that HheA(AD2) has a similar quaternary and tertiary structure but a much more open substrate-binding pocket. Docking experiments reveal that HheA(AD2) can bind both enantiomers of the haloalcohol substrate 1-p-nitrophenyl-2-chloroethanol in a productive way, which explains the low enantiopreference of HheA(AD2). Other differences are found in the halide-binding site, where the side chain amino group of Asn182 is in a position to stabilize the halogen atom or halide ion in HheA(AD2), in contrast to HheC, where a water molecule has taken over this role. These results broaden the insight into the structural determinants that govern reactivity and selectivity in the haloalcohol dehalogenase family.
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http://dx.doi.org/10.1128/JB.01866-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1482898PMC
June 2006

Structural basis for the enantioselectivity of an epoxide ring opening reaction catalyzed by halo alcohol dehalogenase HheC.

J Am Chem Soc 2005 Sep;127(38):13338-43

Department of Biophysical Chemistry, University of Groningen, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands.

Halo alcohol dehalogenase HheC catalyzes the highly enantioselective dehalogenation of vicinal halo alcohols to epoxides, as well as the reverse reaction, the enantioselective and beta-regioselective nucleophilic ring opening of epoxides by pseudo-halides such as azide and cyanide. To investigate this latter reaction, we determined X-ray structures of complexes of HheC with the favored and unfavored enantiomers of para-nitrostyrene oxide. The aromatic parts of the two enantiomers bind in a very similar way, but the epoxide ring of the unfavored (S)-enantiomer binds in a nonproductive inverted manner, with the epoxide oxygen and Cbeta atom positions interchanged with respect to those of the favored (R)-enantiomer. The calculated difference in relative Gibbs binding energy is in agreement with the observed loss of a single hydrogen bond in the S bound state with respect to the R bound state. Our results indicate that it is the nonproductive binding of the unfavored (S)-enantiomer, rather than the difference in affinity for the two enantiomers, that allows HheC to catalyze the azide-mediated ring opening of para-nitrostyrene oxide with high enantioselectivity. This work represents a rare opportunity to explain the enantioselectivity of an enzymatic reaction by comparison of crystallographic data on the binding of both the favored and unfavored enantiomers.
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http://dx.doi.org/10.1021/ja0531733DOI Listing
September 2005

Escherichia coli MltA: MAD phasing and refinement of a tetartohedrally twinned protein crystal structure.

Acta Crystallogr D Biol Crystallogr 2005 May 20;61(Pt 5):613-21. Epub 2005 Apr 20.

Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

Crystals were grown of a mutant form of the bacterial cell-wall maintenance protein MltA that diffracted to 2.15 A resolution. When phasing with molecular replacement using the native structure failed, selenium MAD was used to obtain initial phases. However, after MAD phasing the crystals were found to be tetartohedrally twinned, hampering correct space-group determination and refinement. A refinement protocol was designed to take tetartohedral twinning into account and was successfully applied to refine the structure. The refinement protocol is described and the reasons for the failure of molecular replacement and the success of MAD are discussed in terms of the effects of the tetartohedral twinning.
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http://dx.doi.org/10.1107/S0907444905005743DOI Listing
May 2005

Improved catalytic properties of halohydrin dehalogenase by modification of the halide-binding site.

Biochemistry 2005 May;44(17):6609-18

Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the dehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, resulting in the formation of the corresponding epoxide, a halide ion, and a proton. Halide release is rate-limiting during the catalytic cycle of the conversion of (R)-p-nitro-2-bromo-1-phenylethanol by the enzyme. The recent elucidation of the X-ray structure of HheC showed that hydrogen bonds between the OH group of Tyr187 and between the Odelta1 atom of Asn176 and Nepsilon1 atom of Trp249 could play a role in stabilizing the conformation of the halide-binding site. The possibility that these hydrogen bonds are important for halide binding and release was studied using site-directed mutagenesis. Steady-state kinetic studies revealed that mutant Y187F, which has lost both hydrogen bonds, has a higher catalytic activity (k(cat)) with two of the three tested substrates compared to the wild-type enzyme. Mutant W249F also shows an enhanced k(cat) value with these two substrates, as well as a remarkable increase in enantiopreference for (R)-p-nitro-2-bromo-1-phenylethanol. In case of a mutation at position 176 (N176A and N176D), a 1000-fold lower catalytic efficiency (k(cat)/K(m)) was obtained, which is mainly due to an increase of the K(m) value of the enzyme. Pre-steady-state kinetic studies showed that a burst of product formation precedes the steady state, indicating that halide release is still rate-limiting for mutants Y187F and W249F. Stopped-flow fluorescence experiments revealed that the rate of halide release is 5.6-fold higher for the Y187F mutant than for the wild-type enzyme and even higher for the W249F enzyme. Taken together, these results show that the disruption of two hydrogen bonds around the halide-binding site increases the rate of halide release and can enhance the overall catalytic activity of HheC.
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http://dx.doi.org/10.1021/bi047613zDOI Listing
May 2005

The X-ray structure of trans-3-chloroacrylic acid dehalogenase reveals a novel hydration mechanism in the tautomerase superfamily.

J Biol Chem 2004 Mar 29;279(12):11546-52. Epub 2003 Dec 29.

Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

Isomer-specific 3-chloroacrylic acid dehalogenases function in the bacterial degradation of 1,3-dichloropropene, a compound used in agriculture to kill plant-parasitic nematodes. The crystal structure of the heterohexameric trans-3-chloroacrylic acid dehalogenase (CaaD) from Pseudomonas pavonaceae 170 inactivated by 3-bromopropiolate shows that Glu-52 in the alpha-subunit is positioned to function as the water-activating base for the addition of a hydroxyl group to C-3 of 3-chloroacrylate and 3-bromopropiolate, whereas the nearby Pro-1 in the beta-subunit is positioned to provide a proton to C-2. Two arginine residues, alphaArg-8 and alphaArg-11, interact with the C-1 carboxylate groups, thereby polarizing the alpha,beta-unsaturated acids. The reaction with 3-chloroacrylate results in the production of an unstable halohydrin, 3-chloro-3-hydroxypropanoate, which decomposes into the products malonate semialdehyde and HCl. In the inactivation mechanism, however, malonyl bromide is produced, which irreversibly alkylates the betaPro-1. CaaD is related to 4-oxalocrotonate tautomerase, with which it shares an N-terminal proline. However, in 4-oxalocrotonate tautomerase, Pro-1 functions as a base participating in proton transfer within a hydrophobic active site, whereas in CaaD, the acidic proline is stabilized in a hydrophilic active site. The altered active site environment of CaaD thus facilitates a previously unknown reaction in the tautomerase superfamily, the hydration of the alpha,beta-unsaturated bonds of trans-3-chloroacrylate and 3-bromopropiolate. The mechanism for these hydration reactions represents a novel catalytic strategy that results in carbon-halogen bond cleavage.
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http://dx.doi.org/10.1074/jbc.M311966200DOI Listing
March 2004

Structure and mechanism of bacterial dehalogenases: different ways to cleave a carbon-halogen bond.

Curr Opin Struct Biol 2003 Dec;13(6):722-30

Department of Biophysical Chemistry, University of Groningen, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands.

The dehalogenases make use of fundamentally different strategies to cleave carbon-halogen bonds. The structurally characterized haloalkane dehalogenases, haloacid dehalogenases and 4-chlorobenzoate-coenzyme A dehalogenases use substitution mechanisms that proceed via a covalent aspartyl intermediate. Recent X-ray crystallographic analysis of a haloalcohol dehalogenase and a trans-3-chloroacrylic acid dehalogenase has provided detailed insight into a different intramolecular substitution mechanism and a hydratase-like mechanism, respectively. The available information on the various dehalogenases supports different views on the possible evolutionary origins of their activities.
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http://dx.doi.org/10.1016/j.sbi.2003.10.009DOI Listing
December 2003

Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1.

Acta Crystallogr D Biol Crystallogr 2002 Jan 21;58(Pt 1):176-8. Epub 2001 Dec 21.

Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop vapour-diffusion experiments against 50-70% saturated ammonium sulfate solution at pH 6.5-7.3. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 104.5, c = 121.4 A, and contain two monomers in the asymmetric unit. The crystals diffract to 3.0 A resolution with X-rays from a Cu Kalpha rotating-anode generator.
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http://dx.doi.org/10.1107/s0907444901019618DOI Listing
January 2002