Effects of Removal and Remixing of Heavyweight Pigs on Performance to Slaughter Weights

An experiment was conducted to determine the effects of heavyweight pig removal and remixing on performance. The experiment used a total of 450 pigs (31 kg initial BW) that were sorted and remixed at a mean replicate BW of 73 kg. Treatments were 15 pigs/pen from initial BW to slaughter (15S), 20 pigs/pen from initial BW to time of sort and remix, then reduced to 15 pigs/pen (20/ 15), and 15 pigs/pen from time of sort and remix to slaughter, comprised of the 5 heaviest pigs from each of three 20/15 pens per replicate (15M). Space allocation was 0.56 m2/pig to the day of remixing and 0.74 m2/pig thereafter. There was no effect (P \u3e 0.1) of treatment on ADG before 73 kg BW when pens were 1A contribution of the University of Nebraska Agricultural Research Division, Lincoln, NE 68583. Journal series no. 14679. 2To whom correspondence should be addressed: [email protected] 3Committee members during the study in addition to those listed above were G. Apgar, SIU; M. Carlson, MO; R. K. Christenson, USDA-ARS RLH USMARC; L. Christianson, IL; M. Ellis, IL; R. Goodband, KSU; J. D. Harmon, IA; M. Honeyman, IA; D. D. Jones, IN; S. J. Moeller, OH; B. Richert, IN; K. Stalder, TN; R. C. Thaler, SD. the experimental units. There was no effect (P \u3e 0.1) of treatment on ADG or feed conversion to slaughter BW following removal and remixing using the contrast 20/15 + 15M vs. 15S. The average of the replicate for 20/15 and 15M was used as the experimental unit in a second statistical analysis. Daily feed was less (P = 0.079) from placement to 73 kg BW for the 20/15 + 15M population vs. the 15S population resulting in a lesser (P = 0.067) overall ADG (0.875 vs. 0.887 kg/d, respectively) with no effect (P \u3e 0.1) on feed conversion or CV sample population BW. Removal and remixing of heavyweight pigs at a midpoint in the growth process had minimal effects on performance to slaughter and CV for BW at slaughter

The fully conserved Asp residue in conserved sequence region I of the alpha-amylase family is crucial for the catalytic site architecture and activity

AbstractThe α-amylase family is a large group of starch processing enzymes [Svensson, B. (1994) Plant Mol. Biol. 25, 141–157]. It is characterized by four short sequence motifs that contain the seven fully conserved amino acid residues in this family: two catalytic carboxylic acid residues and four substrate binding residues. The seventh conserved residue (Asp135) has no direct interactions with either substrates or products, but it is hydrogen-bonded to Arg227, which does bind the substrate in the catalytic site. Using cyclodextrin glycosyltransferase as an example, this paper provides for the first time definite biochemical and structural evidence that Asp135 is required for the proper conformation of several catalytic site residues and therefore for activity

The remote substrate binding subsite-6 in cyclodextrin-glycosyltransferase controls the transferase activity of the enzyme via an induced-fit mechanism

Cyclodextrin-glycosyltransferase (CGTase) catalyzes the formation of alpha-, beta-, and gamma-cyclodextrins (cyclic alpha-(1,4)-linked oligosaccharides of 6, 7, or 8 glucose residues, respectively) from starch. Nine substrate binding subsites were observed in an x-ray structure of the CGTase from Bacillus circulans strain 251 complexed with a maltononaose substrate. Subsite -6 is conserved in CGTases, suggesting its importance for the reactions catalyzed by the enzyme. To investigate this in detail, we made six mutant CGTases (Y167F, G179L, G180L, N193G, N193L, and G179L/G180L). All subsite -6 mutants had decreased k(cat) values for beta-cyclodextrin formation, as well as for the disproportionation and coupling reactions, but not for hydrolysis. Especially G179L, G180L, and G179L/G180L affected the transglycosylation activities, most prominently for the coupling reactions. The results demonstrate that (i) subsite -6 is important for all three CGTase-catalyzed transglycosylation reactions, (ii) Gly-180 is conserved because of its importance for the circularization of the linear substrates, (iii) it is possible to independently change cyclization and coupling activities, and (iv) substrate interactions at subsite -6 activate the enzyme in catalysis via an induced-fit mechanism. This article provides for the first time definite biochemical evidence for such an induced-fit mechanism in the alpha-amylase family

Characterization of two bacterial multi-flavinylated proteins harboring multiple covalent flavin cofactors

In recent years, studies have shown that a large number of bacteria secrete multi-flavinylated proteins. The exact roles and properties, of these extracellular flavoproteins that contain multiple covalently anchored FMN cofactors, are still largely unknown. Herein, we describe the biochemical and structural characterization of two multi-FMN-containing covalent flavoproteins, SaFMN3 from Streptomyces azureus and CbFMN4 from Clostridiaceae bacterium. Based on their primary structure, these proteins were predicted to contain three and four covalently tethered FMN cofactors, respectively. The genes encoding SaFMN3 and CbFMN4 were heterologously coexpressed with a flavin transferase (ApbE) in Escherichia coli, and could be purified by affinity chromatography in good yields. Both proteins were found to be soluble and to contain covalently bound FMN molecules. The SaFMN3 protein was studied in more detail and found to display a single redox potential (-184 mV) while harboring three covalently attached flavins. This is in line with the high sequence similarity when the domains of each flavoprotein are compared. The fully reduced form of SaFMN3 is able to use dioxygen as electron acceptor. Single domains from both proteins were expressed, purified and crystallized. The crystal structures were elucidated, which confirmed that the flavin cofactor is covalently attached to a threonine. Comparison of both crystal structures revealed a high similarity, even in the flavin binding pocket. Based on the crystal structure, mutants of the SaFMN3-D2 domain were designed to improve its fluorescence quantum yield by changing the microenvironment of the isoalloxazine moiety of the flavin cofactor. Residues that quench the flavin fluorescence were successfully identified. Our study reveals biochemical details of multi-FMN-containing proteins, contributing to a better understanding of their role in bacteria and providing leads to future utilization of these flavoprotein in biotechnology.</p

Identification and characterization of archaeal and bacterial F420-dependent thioredoxin reductases

The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as reducing cofactor. Yet, in 2016 a new type of thioredoxin reductase was discovered in archaea which utilizes instead a reduced deazaflavin cofactor (F 420 H 2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed these two DFTRs are highly specific for F 420 H 2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that dependent on NADPH (NTRs). A detailed structural analysis led the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR. </p