The Rosetta software suite for macromolecular modeling, docking, and design is widely used in pharmaceutical, industrial, academic, non-profit, and government laboratories. Despite its broad modeling capabilities, Rosetta remains consistently among leading software suites when compared to other methods created for highly specialized protein modeling and design tasks. Developed for over two decades by a global community of over 60 laboratories, Rosetta has undergone multiple refactorings, and now comprises over three million lines of code. Here we discuss methods developed in the last five years in Rosetta, involving the latest protocols for structure prediction; protein-protein and protein-small molecule docking; protein structure and interface design; loop modeling; the incorporation of various types of experimental data; modeling of peptides, antibodies and proteins in the immune system, nucleic acids, non-standard chemistries, carbohydrates, and membrane proteins. We briefly discuss improvements to the energy function, user interfaces, and usability of the software. Rosetta is available at www.rosettacommons.org.

Haloalcohol dehalogenases are bacterial enzymes that catalyze the cofactor-independent dehalogenation of vicinal haloalcohols such as the genotoxic environmental pollutant 1,3-dichloro-2-propanol, thereby producing an epoxide, a chloride ion and a proton. Here we present X-ray structures of the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1, and complexes of the enzyme with an epoxide product and chloride ion, and with a bound haloalcohol substrate mimic. These structures support a catalytic mechanism in which Tyr145 of a Ser-Tyr-Arg catalytic triad deprotonates the haloalcohol hydroxyl function to generate an intramolecular nucleophile that substitutes the vicinal halogen. Haloalcohol dehalogenases are related to the widespread family of NAD(P)Hdependent short-chain dehydrogenases/reductases (SDR family), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze reductive or oxidative conversions of various secondary alcohols and ketones. Our results reveal the ®rst structural details of an SDR-related enzyme that catalyzes a substitutive dehalogenation reaction rather than a redox reaction, in which a halide-binding site is found at the location of the NAD(P)H binding site. Structure-based sequence analysis reveals that the various haloalcohol dehalogenases have likely originated from at least two different NAD-binding SDR precursors. Keywords: haloalcohol dehalogenase/SDR family/shortchain dehydrogenase/X-ray structure IntroductionDehalogenases are enzymes that are able to cleave carbonhalogen bonds . Structural characterization of haloalkane dehalogenases and haloacid dehalogenases demonstrated that these hydrolytic enzymes are evolutionarily related to widespread esterase and phosphatase families (Ollis et al., 1992;Hisano et al., 1996;Ridder and Dijkstra, 1999). Haloalcohol dehalogenases, also known as halohydrin dehalogenases or halohydrin hydrogen-halide lyases, cannot be classi®ed in these existing dehalogenase families. Instead, they show low sequence similarity to members of the short-chain dehydrogenase/reductase (SDR) family . This family contains redox enzymes that depend on NAD(P)H, which is bound in a characteristic dinucleotide binding fold (Rossmann fold) (Rossmann et al., 1974). They have a conserved catalytic triad of Ser, Tyr and Lys/Arg residues (Jo Èrnvall et al., 1995;Oppermann et al., 2003), which is also present in the haloalcohol dehalogenases . Many structures of shortchain dehydrogenases/reductases in complex with dinucleotides and substrates have revealed the structural details of the reactions catalyzed by them (Jo Èrnvall et al., 1995;Filling et al., 2002;Oppermann et al., 2003). In addition, the structure of a dinucleotide-binding transcription factor that lacked the catalytic tyrosine and thus oxidoreductase activity showed that the SDR fold also functions in nonenzymatic activities (Stammers et al., 2001).Haloalcohol dehalogenases catalyze the intramolecular displacement of a halogen by the vicinal hydroxyl group in 1,3-dichloro-2-propanol, yielding its ...

Summary 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.

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 ␣-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 ␤-subunit is positioned to provide a proton to C-2. Two arginine residues, ␣Arg-8 and ␣Arg-11, interact with the C-1 carboxylate groups, thereby polarizing the ␣,␤-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 ␤Pro-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 ␣,␤-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.Dehalogenases are enzymes that cleave carbon-halogen bonds. They are found in various bacteria, allowing them to use halogenated hydrocarbons as growth substrates (1, 2). Detailed three-dimensional structural information is available for dehalogenases such as haloalkane dehalogenase, 2-haloacid dehalogenase, and haloalcohol dehalogenase, which catalyze the cofactor-independent cleavage of the covalent bond between a halogen and an sp 3 -hybridized carbon atom by substitution mechanisms (3-5). In addition, several cofactor-dependent dehalogenases have been characterized that cleave the bond between a halogen and an sp 2 -hybridized carbon atom. Examples include heme-dependent reductive dehalogenases (6, 7) and the 4-chlorobenzoyl-CoA dehalogenases (8). In contrast, cofactorindependent dehalogenases that cleave the bond between a halogen and an sp 2 -hybridized carbon atom have only recently been discovered (9, 10).The 3-chloroacrylic acid dehalogenases from Pseudomonas pavonaceae 170 represent cofactor-independent dehalogenases that catalyze the cleavage of vinylic carbon-halogen bonds, in which the halogen is bound to an sp 2 -hybridized carbon atom (9 -11). They are part of a multienzyme degradation route for the cis-and trans-isomers of 1,3-dichloropropene (DCP).1 Cisand trans-DCP are components of the commercially produced fumigants Shell D-D and Telone II, which are us...

BackgroundEngineering 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.ResultsWe 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.ConclusionsEngineering 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0168-9) contains supplementary material, which is available to authorized users.