An Isoprene Lipid-Binding Protein Promotes Eukaryotic Coenzyme Q Biosynthesis.

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. In eukaryotes, CoQ precursors-among nature's most hydrophobic molecules-must somehow be presented to a series of enzymes peripherally associated with the mitochondrial inner membrane. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. We show that COQ9 repurposes the bacterial TetR fold to bind aromatic isoprenes with high specificity, including CoQ intermediates that likely reside entirely within the bilayer. We reveal a process by which COQ9 associates with cardiolipin-rich membranes and warps the membrane surface to access this cargo. Finally, we identify a molecular interface between COQ9 and the hydroxylase COQ7, motivating a model whereby COQ9 presents intermediates directly to CoQ enzymes. Overall, our results provide a mechanism for how a lipid-binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.

Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.

Quantitative Analysis of The High-Yield Hydrolysis of Kelp by Laminarinase and Alginate Lyase.

Kelp is an abundant, farmable biomass-containing laminarin and alginate as major polysaccharides, providing an excellent model substrate to study their deconstruction by simple enzyme mixtures. Our previous study showed strong reactivity of the glycoside hydrolase family 55 during hydrolysis of purified laminarin, raising the question of its reactivity with intact kelp. In this study, we determined that a combination of a single glycoside hydrolase family 55 ß-1,3-exoglucanase with a broad-specificity alginate lyase from the polysaccharide lyase family 18 gives efficient hydrolysis of untreated kelp to a mixture of simple sugars, that is, glucose, gentiobiose, mannitol-end glucose, and mannuronic and guluronic acids and their soluble oligomers. Quantitative assignments from nanostructure initiator mass spectrometry (NIMS) and 2D HSQC NMR spectroscopy and analysis of the reaction time-course are provided. The data suggest that binary combinations of enzymes targeted to the unique polysaccharide composition of marine biomass are sufficient to deconstruct kelp into soluble sugars for microbial fermentation.

Identification and characterization of a set of monocot BAHD monolignol transferases.

Plant BAHD acyltransferases perform a wide range of enzymatic tasks in primary and secondary metabolism. Acyl-CoA monolignol transferases, which couple a CoA substrate to a monolignol creating an ester linkage, represent a more recent class of such acyltransferases. The resulting conjugates may be used for plant defense but are also deployed as important "monomers" for lignification, in which they are incorporated into the growing lignin polymer chain. p-Coumaroyl-CoA monolignol transferases (PMTs) increase the production of monolignol p-coumarates, and feruloyl-CoA monolignol transferases (FMTs) catalyze the production of monolignol ferulate conjugates. We identified putative FMT and PMT enzymes in sorghum (Sorghum bicolor) and switchgrass (Panicum virgatum) and have compared their activities to those of known monolignol transferases. The putative FMT enzymes produced both monolignol ferulate and monolignol p-coumarate conjugates, whereas the putative PMT enzymes produced monolignol p-coumarate conjugates. Enzyme activity measurements revealed that the putative FMT enzymes are not as efficient as the rice (Oryza sativa) control OsFMT enzyme under the conditions tested, but the SbPMT enzyme is as active as the control OsPMT enzyme. These putative FMTs and PMTs were transformed into Arabidopsis (Arabidopsis thaliana) to test their activities and abilities to biosynthesize monolignol conjugates for lignification in planta. The presence of ferulates and p-coumarates on the lignin of these transformants indicated that the putative FMTs and PMTs act as functional feruloyl-CoA and p-coumaroyl-CoA monolignol transferases within plants.

pHBMT1, a BAHD-family monolignol acyltransferase, mediates lignin acylation in poplar.

Poplar (Populus) lignin is naturally acylated with p-hydroxybenzoate ester moieties. However, the enzyme(s) involved in the biosynthesis of the monolignol-p-hydroxybenzoates have remained largely unknown. Here, we performed an in vitro screen of the Populus trichocarpa BAHD acyltransferase superfamily (116 genes) using a wheatgerm cell-free translation system and found five enzymes capable of producing monolignol-p-hydroxybenzoates. We then compared the transcript abundance of the five corresponding genes with p-hydroxybenzoate concentrations using naturally occurring unrelated genotypes of P. trichocarpa and revealed a positive correlation between the expression of p-hydroxybenzoyl-CoA monolig-nol transferase (pHBMT1, Potri.001G448000) and p-hydroxybenzoate levels. To test whether pHBMT1 is responsible for the biosynthesis of monolignol-p-hydroxybenzoates, we overexpressed pHBMT1 in hybrid poplar (Populus alba × P. grandidentata) (35S::pHBMT1 and C4H::pHBMT1). Using three complementary analytical methods, we showed that there was an increase in soluble monolignol-p-hydroxybenzoates and cell-wall-bound monolignol-p-hydroxybenzoates in the poplar transgenics. As these pendent groups are ester-linked, saponification releases p-hydroxybenzoate, a precursor to parabens that are used in pharmaceuticals and cosmetics. This identified gene could therefore be used to engineer lignocellulosic biomass with increased value for emerging biorefinery strategies.

A bacterial biosynthetic pathway for methylated furan fatty acids.

Fatty acids play many important roles in cells and also in industrial processes. Furan fatty acids (FuFAs) are present in the lipids of some plant, fish, and microbial species and appear to function as second messengers in pathways that protect cells from membrane-damaging agents. We report here the results of chemical, genetic, and synthetic biology experiments to decipher the biosynthesis of the monomethylated FuFA, methyl 9-(3-methyl-5-pentylfuran-2-yl) nonanoate (9M5-FuFA), and its dimethyl counterpart, methyl 9-(3,4-dimethyl-5-pentylfuran-2-yl) nonanoate (9D5-FuFA), in two α-proteobacteria. Each of the steps in FuFA biosynthesis occurs on pre-existing phospholipid fatty acid chains, and we identified pathway intermediates and the gene products that catalyze 9M5-FuFA and 9D5-FuFA synthesis in Rhodobacter sphaeroides 2.4.1 and Rhodopseudomonas palustris CGA009. One previously unknown pathway intermediate was a methylated diunsaturated fatty acid, (10E,12E)-11-methyloctadeca-10,12-dienoic acid (11Me-10t,12t-18:2), produced from (11E)-methyloctadeca-11-enoic acid (11Me-12t-18:1) by a newly identified fatty acid desaturase, UfaD. We also show that molecular oxygen (O2) is the source of the oxygen atom in the furan ring of 9M5-FuFA, and our findings predict that an O2-derived oxygen atom is incorporated into 9M5-FuFA via a protein, UfaO, that uses the 11Me-10t,12t-18:2 fatty acid phospholipid chain as a substrate. We discovered that R. palustris also contains a SAM-dependent methylase, FufM, that produces 9D5-FuFA from 9M5-FuFA. These results uncover the biochemical sequence of intermediates in a bacterial pathway for 9M5-FuFA and 9D5-FuFA biosynthesis and suggest the existence of homologs of the enzymes identified here that could function in FuFA biosynthesis in other organisms.

Mannose- and Mannobiose-Specific Responses of the Insect-Associated Cellulolytic Bacterium Streptomyces sp. Strain SirexAA-E.

The cellulolytic insect symbiont bacterium Streptomyces sp. strain SirexAA-E secretes a suite of carbohydrate-active enzymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. Using genomic analysis, we found a unique 12-bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and thus they are likely under the regulation of both SsManR and SsCebR. IMPORTANCEStreptomyces sp. SirexAA-E, a microbial symbiont of biomass-harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.

A heterodimeric glutathione S-transferase that stereospecifically breaks lignin's ß(R)-aryl ether bond reveals the diversity of bacterial ß-etherases.

Lignin is a heterogeneous polymer of aromatic subunits that is a major component of lignocellulosic plant biomass. Understanding how microorganisms deconstruct lignin is important for understanding the global carbon cycle and could aid in developing systems for processing plant biomass into valuable commodities. Sphingomonad bacteria use stereospecific glutathione S-transferases (GSTs) called ß-etherases to cleave the ß-aryl ether (ß-O-4) bond, the most common bond between aromatic subunits in lignin. Previously characterized bacterial ß-etherases are homodimers that fall into two distinct GST subclasses: LigE homologues, which cleave the ß(R) stereoisomer of the bond, and LigF homologues, which cleave the ß(S) stereoisomer. Here, we report on a heterodimeric ß-etherase (BaeAB) from the sphingomonad Novosphingobium aromaticivorans that stereospecifically cleaves the ß(R)-aryl ether bond of the di-aromatic compound ß-(2-methoxyphenoxy)-γ-hydroxypropiovanillone (MPHPV). BaeAB's subunits are phylogenetically distinct from each other and from other ß-etherases, although they are evolutionarily related to LigF, despite the fact that BaeAB and LigF cleave different ß-aryl ether bond stereoisomers. We identify amino acid residues in BaeAB's BaeA subunit important for substrate binding and catalysis, including an asparagine that is proposed to activate the GSH cofactor. We also show that BaeAB homologues from other sphingomonads can cleave ß(R)-MPHPV and that they may be as common in bacteria as LigE homologues. Our results suggest that the ability to cleave the ß-aryl ether bond arose independently at least twice in GSTs and that BaeAB homologues may be important for cleaving the ß(R)-aryl ether bonds of lignin-derived oligomers in nature.

The novel dual glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 (GLP-1) receptor agonist tirzepatide transiently delays gastric emptying similarly to selective long-acting GLP-1 receptor agonists.

The effect of dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist (RA) tirzepatide on gastric emptying (GE) was compared to that of GLP-1RAs in non-clinical and clinical studies. GE was assessed following acute and chronic treatment with tirzepatide in diet-induced obese mice versus semaglutide or long-acting GIP analogue alone. Participants [with and without type 2 diabetes (T2DM)] from a phase 1, 4-week multiple dose study received tirzepatide, dulaglutide or placebo. GE was assessed by acetaminophen absorption. In mice, tirzepatide delayed GE to a similar degree to that achieved with semaglutide; however, these acute inhibitory effects were abolished after 2 weeks of treatment. GIP analogue alone had no effect on GE or on GLP-1's effect on GE. In participants with and without T2DM, once-weekly tirzepatide (≥5 and ≥4.5 mg, respectively) delayed GE after a single dose. This effect diminished after multiple doses of tirzepatide or dulaglutide in healthy participants. In participants with T2DM treated with an escalation schedule of tirzepatide 5/5/10/10 or 5/5/10/15 mg, a residual GE delay was still observed after multiple doses. These data suggest that tirzepatide's activity on GE is comparable to that of selective GLP-1RAs.

A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.

Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis.

Coenzyme Q (CoQ) is an isoprenylated quinone that is essential for cellular respiration and is synthesized in mitochondria by the combined action of at least nine proteins (COQ1-9). Although most COQ proteins are known to catalyze modifications to CoQ precursors, the biochemical role of COQ9 remains unclear. Here, we report that a disease-related COQ9 mutation leads to extensive disruption of the CoQ protein biosynthetic complex in a mouse model, and that COQ9 specifically interacts with COQ7 through a series of conserved residues. Toward understanding how COQ9 can perform these functions, we solved the crystal structure of Homo sapiens COQ9 at 2.4 Å. Unexpectedly, our structure reveals that COQ9 has structural homology to the TFR family of bacterial transcriptional regulators, but that it adopts an atypical TFR dimer orientation and is not predicted to bind DNA. Our structure also reveals a lipid-binding site, and mass spectrometry-based analyses of purified COQ9 demonstrate that it associates with multiple lipid species, including CoQ itself. The conserved COQ9 residues necessary for its interaction with COQ7 comprise a surface patch around the lipid-binding site, suggesting that COQ9 might serve to present its bound lipid to COQ7. Collectively, our data define COQ9 as the first, to our knowledge, mammalian TFR structural homolog and suggest that its lipid-binding capacity and association with COQ7 are key features for enabling CoQ biosynthesis.

Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein.

Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.

Tirzepatide modulates the regulation of adipocyte nutrient metabolism through long-acting activation of the GIP receptor.

Tirzepatide, a glucose-dependent insulinotropic polypeptide/glucagon-like peptide 1 receptor (GIPR/GLP-1R) agonist, has, in clinical trials, demonstrated greater reductions in glucose, body weight, and triglyceride levels compared with selective GLP-1R agonists in people with type 2 diabetes (T2D). However, cellular mechanisms by which GIPR agonism may contribute to these improved efficacy outcomes have not been fully defined. Using human adipocyte and mouse models, we investigated how long-acting GIPR agonists regulate fasted and fed adipocyte functions. In functional assays, GIPR agonism enhanced insulin signaling, augmented glucose uptake, and increased the conversion of glucose to glycerol in a cooperative manner with insulin; however, in the absence of insulin, GIPR agonists increased lipolysis. In diet-induced obese mice treated with a long-acting GIPR agonist, circulating triglyceride levels were reduced during oral lipid challenge, and lipoprotein-derived fatty acid uptake into adipose tissue was increased. Our findings support a model for long-acting GIPR agonists to modulate both fasted and fed adipose tissue function differentially by cooperating with insulin to augment glucose and lipid clearance in the fed state while enhancing lipid release when insulin levels are reduced in the fasted state.

Development of a long-acting relaxin analogue, LY3540378, for treatment of chronic heart failure.

BACKGROUND AND PURPOSE: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure. EXPERIMENTAL APPROACH: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain. KEY RESULTS: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin. The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats. In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric relaxation time. In a monkey cardiovascular safety study, there were no adverse observations from administration of LY3540378. CONCLUSION AND IMPLICATIONS: LY3540378 demonstrated to be a suitable clinical development candidate, and is progressing in clinical trials.

The cytokine GDF15 signals through a population of brainstem cholecystokinin neurons to mediate anorectic signalling.

The cytokine, GDF15, is produced in pathological states which cause cellular stress, including cancer. When over expressed, it causes dramatic weight reduction, suggesting a role in disease-related anorexia. Here, we demonstrate that the GDF15 receptor, GFRAL, is located in a subset of cholecystokinin neurons which span the area postrema and the nucleus of the tractus solitarius of the mouse. GDF15 activates GFRALAP/NTS neurons and supports conditioned taste and place aversions, while the anorexia it causes can be blocked by a monoclonal antibody directed at GFRAL or by disrupting CCK neuronal signalling. The cancer-therapeutic drug, cisplatin, induces the release of GDF15 and activates GFRALAP/NTS neurons, as well as causing significant reductions in food intake and body weight in mice. These metabolic effects of cisplatin are abolished by pre-treatment with the GFRAL monoclonal antibody. Our results suggest that GFRAL neutralising antibodies or antagonists may provide a co-treatment opportunity for patients undergoing chemotherapy.

Long-Acting and Selective Oxytocin Peptide Analogs Show Antidiabetic and Antiobesity Effects in Male Mice.

Oxytocin (OXT) has been shown to suppress appetite, induce weight loss, and improve glycemic control and lipid metabolism in several species, including humans, monkeys, and rodents. However, OXT's short half-life in circulation and lack of receptor selectivity limit its application and efficacy. In this study, we report an OXT peptide analog (OXTGly) that is potent and selective for the OXT receptor (OXTR). OXT, but not OXTGly, activated vasopressin receptors in vitro and acutely increased blood pressure in vivo when administered IP. OXT suppressed food intake in mice, whereas OXTGly had a moderate effect on food intake when administered IP or intracerebroventricularly. Both OXT (IP) and OXTGly (IP) improved glycemic control in glucose tolerance tests. Additionally, both OXT (IP) and OXTGly (IP) stimulated insulin, glucagon-like peptide 1, and glucagon secretion in mice. We generated lipid-conjugated OXT (acylated-OXT) and OXTGly (acylated-OXTGly) and demonstrated that these molecules have significantly extended half-lives in vivo. Compared with OXT, 2-week treatment of diet-induced obese mice with acylated-OXT [subcutaneous(ly) (SC)] resulted in enhanced body weight reduction, an improved lipid profile, and gene expression changes consistent with increased lipolysis and decreased gluconeogenesis. Treatment with acylated-OXTGly (SC) also resulted in a statistically significant weight loss, albeit to a lesser degree compared with acylated-OXT treatment. In conclusion, we demonstrate that selective activation of the OXTR pathway results in both acute and chronic metabolic benefits, whereas potential activation of vasopressin receptors by nonselective OXT analogs causes physiological stress that contributes to additional weight loss.

Determination of glycoside hydrolase specificities during hydrolysis of plant cell walls using glycome profiling.

BACKGROUND: Glycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions. RESULTS: Single, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss of specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose- and xylose-backbone polysaccharides. CONCLUSIONS: The identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oxime-NIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass.

Automated cell-free protein production methods for structural studies.

In contrast to cell-based protein expression, cell-free production is highly consistent, scalable, and amenable to automation. Robots can handle many samples and perform repetitive procedures that are otherwise prone to human error. Here is described commercially available robotics for a wheat germ cell-free system with emphasis on practical applications for structural and functional studies. In addition, described is a cell-free method for preparing protein complexes.

Cell-free protein synthesis for functional and structural studies.

Recent advances in cell-free protein expression systems have made them reliable and practical for functional and structural studies of a wide variety of proteins. In particular, wheat germ cell-free translation can consistently produce target proteins in microgram quantities from relatively inexpensive, small-scale reactions. Here we describe our small-scale protein expression method for rapidly producing proteins for functional assay and techniques for determining if the target is suitable for scale-up to amounts potentially needed for structure determination. The cell-free system is versatile and can be easily customized with the inclusion of additives. We describe simple modifications used for producing membrane proteins.