Christopher M. Bianchetti

Adaptive evolution of threonine deaminase in plant defense against insect herbivores

Gene duplication is a major source of plant chemical diversity that mediates plant–herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to α-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.

Comparison of Apo- and Heme-bound Crystal Structures of a Truncated Human Heme Oxygenase-2

Heme oxygenase (HO) catalyzes the first step in the heme degradation pathway. The crystal structures of apo- and heme-bound truncated human HO-2 reveal a primarily α-helical architecture similar to that of human HO-1 and other known HOs. Proper orientation of heme in HO-2 is required for the regioselective oxidation of the α-mesocarbon. This is accomplished by interactions within the heme binding pocket, which is made up of two helices. The iron coordinating residue, His45, resides on the proximal helix. The distal helix contains highly conserved glycine residues that allow the helix to flex and interact with the bound heme. Tyr154, Lys199, and Arg203 orient the heme through direct interactions with the heme propionates. The rearrangements of side chains in heme-bound HO-2 compared with apoHO-2 further elucidate HO-2 heme interactions.

The structure and NO binding properties of the nitrophorin‐like heme‐binding protein from Arabidopsis thaliana gene locus At1g79260.1

The protein from Arabidopsis thaliana gene locus At1g79260.1 is comprised of 166‐residues and is of previously unknown function. Initial structural studies by the Center for Eukaryotic Structural Genomics (CESG) suggested that this protein might bind heme, and consequently, the crystal structures of apo and heme‐bound forms were solved to near atomic resolution of 1.32 Å and 1.36 Å, respectively. The rate of hemin loss from the protein was measured to be 3.6 × 10−5 s−1, demonstrating that it binds heme specifically and with high affinity. The protein forms a compact 10‐stranded β‐barrel that is structurally similar to the lipocalins and fatty acid binding proteins (FABPs). One group of lipocalins, the nitrophorins (NP), are heme proteins involved in nitric oxide (NO) transport and show both sequence and structural similarity to the protein from At1g79260.1 and two human homologues, all of which contain a proximal histidine capable of coordinating a heme iron. Rapid‐mixing and laser photolysis techniques were used to determine the rate constants for carbon monoxide (CO) binding to the ferrous form of the protein (k′CO = 0.23 μM−1 s−1, kCO = 0.050 s−1) and NO binding to the ferric form (k′NO = 1.2 μM–1 s–1, kNO = 73 s−1). Based on both structural and functional similarity to the nitrophorins, we have named the protein nitrobindin and hypothesized that it plays a role in NO transport. However, one of the two human homologs of nitrobindin contains a THAP domain, implying a possible role in apoptosis. Proteins 2010.

Structure, dynamics, and specificity of endoglucanase D from Clostridium cellulovorans.

The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the β-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-Å-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trp162 plays a direct role in substrate binding.

Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

BackgroundCarbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridiumthermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed.ResultsCelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass.ConclusionWe have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.

Fusion of Dioxygenase and Lignin-binding Domains in a Novel Secreted Enzyme from Cellulolytic Streptomyces sp. SirexAA-E.

Background: Fusions of dioxygenase and CBMs have been predicted in cellulolytic microbes. Results: SACTE_2871 is unique two-domain enzyme that reacts with caffeoyl-CoA and shows preferential binding to synthetic lignins. Conclusion: SACTE_2871 is an intradiol dioxygenase that is targeted to growing surfaces of lignin. Significance: SACTE_2871 can destroy precursors needed by the plant for de novo lignin biosynthesis as part of its natural wounding response. Streptomyces sp. SirexAA-E is a highly cellulolytic bacterium isolated from an insect/microbe symbiotic community. When grown on lignin-containing biomass, it secretes SACTE_2871, an aromatic ring dioxygenase domain fused to a family 5/12 carbohydrate-binding module (CBM 5/12). Here we present structural and catalytic studies of this novel fusion enzyme, thus providing insight into its function. The dioxygenase domain has the core β-sandwich fold typical of this enzyme family but lacks a dimerization domain observed in other intradiol dioxygenases. Consequently, the x-ray structure shows that the enzyme is monomeric and the Fe(III)-containing active site is exposed to solvent in a shallow depression on a planar surface. Purified SACTE_2871 catalyzes the O2-dependent intradiol cleavage of catechyl compounds from lignin biosynthetic pathways, but not their methylated derivatives. Binding studies show that SACTE_2871 binds synthetic lignin polymers and chitin through the interactions of the CBM 5/12 domain, representing a new binding specificity for this fold-family. Based on its unique structural features and functional properties, we propose that SACTE_2871 contributes to the invasive nature of the insect/microbial community by destroying precursors needed by the plant for de novo lignin biosynthesis as part of its natural wounding response.

Active Site and Laminarin Binding in Glycoside Hydrolase Family 55

Background: SacteLam55A is a GH55 enzyme from highly cellulolytic Streptomyces sp. SirexAA-E. Results: Substrate-bound structures identify residues involved in binding, catalysis, enforcement of reaction specificity, and possibly processivity. Conclusion: Natural GH55 are exo-β-1,3-glucanases with a broad range of temperature and pH optima. Significance: Experimental annotation of GH phylogenetic space by use of bioinformatics, high throughput cell-free translation, biochemical assay, and structure determination is feasible. The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.

Structure of cellobiose phosphorylase from Clostridium thermocellum in complex with phosphate

Clostridium thermocellum is a cellulosome-producing bacterium that is able to efficiently degrade and utilize cellulose as a sole carbon source. Cellobiose phosphorylase (CBP) plays a critical role in cellulose degradation by catalyzing the reversible phosphate-dependent hydrolysis of cellobiose, the major product of cellulose degradation, into α-D-glucose 1-phosphate and D-glucose. CBP from C. thermocellum is a modular enzyme composed of four domains [N-terminal domain, helical linker, (α/α)(6)-barrel domain and C-terminal domain] and is a member of glycoside hydrolase family 94. The 2.4 Å resolution X-ray crystal structure of C. thermocellum CBP reveals the residues involved in coordinating the catalytic phosphate as well as the residues that are likely to be involved in substrate binding and discrimination.

Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles

Ribonuclease inhibitor (RI) is a conserved protein of the mammalian cytosol. RI binds with high affinity to diverse secretory ribonucleases (RNases) and inhibits their enzymatic activity. Although secretory RNases are found in all vertebrates, the existence of a non-mammalian RI has been uncertain. Here, we report on the identification and characterization of RI homologs from chicken and anole lizard. These proteins bind to RNases from multiple species but exhibit much greater affinity for their cognate RNases than for mammalian RNases. To reveal the basis for this differential affinity, we determined the crystal structure of mouse, bovine, and chicken RI·RNase complexes to a resolution of 2.20, 2.21, and 1.92Å, respectively. A combination of structural, computational, and bioinformatic analyses enabled the identification of two residues that appear to contribute to the differential affinity for RNases. We also found marked differences in oxidative instability between mammalian and non-mammalian RIs, indicating evolution toward greater oxygen sensitivity in RIs from mammalian species. Taken together, our results illuminate the structural and functional evolution of RI, along with its dynamic role in vertebrate biology.

Structure-guided analysis of catalytic specificity of the abundantly secreted chitosanase SACTE_5457 from Streptomyces sp. SirexAA-E

SACTE_5457 is secreted by Streptomyces sp. SirexAA‐E, a highly cellulolytic actinobacterium isolated from a symbiotic community composed of insects, fungi, and bacteria. Here we report the 1.84 Å resolution crystal structure and functional characterization of SACTE_5457. This enzyme is a member of the glycosyl hydrolase family 46 and is composed of two α‐helical domains that are connected by an α‐helical linker. The catalytic residues (Glu74 and Asp92) are separated by 10.3 Å, matching the distance predicted for an inverting hydrolysis reaction. Normal mode analysis suggests that the connecting α‐helix is flexible and allows the domain motion needed to place active site residues into an appropriate configuration for catalysis. SACTE_5457 does not react with chitin, but hydrolyzes chitosan substrates with an ∼4‐fold improvement in kcat/KM as the percentage of acetylation and the molecular weights decrease. Analysis of the time dependence of product formation shows that oligosaccharides with degree of polymerization <4 are not hydrolyzed. By combining the results of substrate docking to the X‐ray structure and end‐product analysis, we deduce that SACTE_5457 preferentially binds substrates spanning the −2 to +2 sugar binding subsites, and that steric hindrance prevents binding of N‐acetyl‐d‐glucosamine in the +2 subsite and may weakly interfere with binding of N‐acetyl‐d‐glucosamine in the +1 subsites. A proposal for how these constraints account for the observed product distributions is provided. Proteins 2014; 82:1245–1257.