Impact of crossbridge structure on peptidoglycan crosslinking: A synthetic stem peptide approach.

The bacterial cell wall, whose main component is peptidoglycan (PG), provides cellular rigidity and prevents lysis from osmotic pressure. Moreover, the cell wall is the main interface between the external environment and internal cellular components. Given its essentiality, many antibiotics target enzymes related to the biosynthesis of cell wall. Of these enzymes, transpeptidases (TPs) are central to proper cell wall assembly and their inactivation is the mechanism of action of many antibiotics including ß-lactams. TPs are responsible for stitching together strands of PG to make the crosslinked meshwork of the cell wall. This chapter focuses on the use of solid-phase peptide synthesis to build PG analogs that become site-selectively incorporated into the cell wall of live bacterial cells. This method allows for the design of fluorescent handles on PG probes that will enable the interrogation of substrate preferences of TPs (e.g., amidation at the glutamic acid residue, crossbridge presence) by analyzing the level of probe incorporation within the native cell wall of live bacterial cells.

Effects of enzymatic free fatty acid reduction process on the composition and phytochemicals of rice bran oil.

The effects of enzymatic free fatty acid reduction process (EFFARP) on the composition and phytochemicals of dewaxed and degummed rice bran oil (DDRBO) were investigated and compared with the effects observed using internal acyl acceptors. The acid value of DDRBO was effectively decreased from 16.99 mg KOH/g to approximately 0.36 mg KOH/g by EFFARP. EFFARP significantly decreased the moisture content and peroxide value of DDRBO and increased the induction period. The Sn-2 fatty acid comoposition of DDRBO after EFFARP was very reaching the total fatty acid composition. EFFARP significantly increased the triacylglycerol content compared to the control, while the oryzanol content was not obviously affected. The contents of free sterol, and total tocopherol and tocotrienol were increased slightly by EFFARP compared to the control. When conducted under vacuum with added nitrogen, EFFARP shows great application potential in the edible oil industry.

Structural and Biochemical Insights Into Two BAHD Acyltransferases (AtSHT and AtSDT) Involved in Phenolamide Biosynthesis.

Phenolamides represent one of the largest classes of plant-specialized secondary metabolites and function in diverse physiological processes, including defense responses and development. The biosynthesis of phenolamides requires the BAHD-family acyltransferases, which transfer acyl-groups from different acyl-donors specifically to amines, the acyl-group acceptors. However, the mechanisms of substrate specificity and multisite-acylation of the BAHD-family acyltransferases remain poorly understood. In this study, we provide a structural and biochemical analysis of AtSHT and AtSDT, two representative BAHD-family members that catalyze the multisite acylation of spermidine but show different product profiles. By determining the structures of AtSHT and AtSDT and using structure-based mutagenesis, we identified the residues important for substrate recognition in AtSHT and AtSDT and hypothesized that the acyl acceptor spermidine might adopt a free-rotating conformation in AtSHT, which can undergo mono-, di-, or tri-acylation; while the spermidine molecule in AtSDT might adopt a linear conformation, which only allows mono- or di-acylation to take place. In addition, through sequence similarity network (SSN) and structural modeling analysis, we successfully predicted and verified the functions of two uncharacterized Arabidopsis BAHD acyltransferases, OAO95042.1 and NP_190301.2, which use putrescine as the main acyl-acceptor. Our work provides not only an excellent starting point for understanding multisite acylation in BAHD-family enzymes, but also a feasible methodology for predicting possible acyl acceptor specificity of uncharacterized BAHD-family acyltransferases.

Expanding of Phospholipid:Diacylglycerol AcylTransferase (PDAT) from Saccharomyces cerevisiae as Multifunctional Biocatalyst with Broad Acyl Donor/Acceptor Selectivity.

Triacylglycerols are considered one of the most promising feedstocks for biofuels. Phospholipid:diacylglycerol acyltransferase (PDAT), responsible for the last step of triacylglycerol synthesis in the acyl-CoA-independent pathway, has attracted much attention by catalyzing membrane lipid transformation. However, due to lack of biochemical and enzymatic studies, PDAT has not carried forward in biocatalyst application. Here, the PDAT from Saccharomyces cerevisiae was expressed in Pichia pastoris. The purified enzymes were studied using different acyl donors and acceptors by thin layer chromatography and gas chromatography. In addition of the preferred acyl donor of PE and PC, the results identified that ScPDAT was capable of using broad acyl donors such as PA, PS, PG, MGDG, DGDG, and acyl-CoA, and ScPDAT was more likely to use unsaturated acyl donors comparing 18:0/18:1 to 18:0/18:0 phospholipids. With regard to acyl acceptors, ScPDAT preferred 1,2 to 1,3-diacylglycerol (DAG), while 12:0/12:0 DAG was identified as the optimal acyl acceptor, followed by 18:1/18:1 and 18:1/16:0 DAG. Additionally, ScPDAT reveals esterification activity that can utilize methanol as acyl acceptor to generate fatty acid methyl esters. The results fully expand the enzymatic selectivity of ScPDAT and provide fundamental knowledge for synthesis of triacylglycerol-derived biofuels.

Efficient Production of Homogeneous Lysine-Based Antibody Conjugates Using Microbial Transglutaminase.

Random conjugation of chemical linkers to endogenous lysines or cysteines within an antibody yields a heterogeneous mixture of conjugates with various drug-to-antibody ratios. One approach for generating homogeneous antibody conjugates utilizes enzymatic transfer of payloads onto a specific glycan or amino acid residue. Microbial transglutaminase (MTG) is an enzyme that catalyzes the formation of a stable isopeptide bond between a glutamine and a lysine. We have previously identified and reported several sites throughout the antibody structure where an engineered lysine is sufficient for transfer of a glutamine-based substrate onto the antibody. Whereas other enzymatic transfer strategies typically require significant antibody engineering to either modify the N-glycans or introduce a multi-amino acid enzyme recognition site, the lower contextual specificity of MTG for lysines allows just a single lysine point mutation in an antibody to be efficiently transamidated. Here we describe the molecular positioning of these single engineered lysine residues and the conjugation conditions for producing homogeneous antibody conjugates exemplified using azido- and auristatin F-based acyl donor substrates.

Abrogation of Immunogenic Properties of Gliadin Peptides through Transamidation by Microbial Transglutaminase Is Acyl-Acceptor Dependent.

Wheat gluten confers superior baking quality to wheat based products but elicits a pro-inflammatory immune response in patients with celiac disease. Transamidation of gluten by microbial transglutaminase (mTG) and tissue transglutaminase (tTG) reduces the immunogenicity of gluten; however, little information is available on the minimal modification sufficient to eliminate gliadin immunogenicity nor has the effectiveness of transamidation been studied with T-cell clones from patients. Here we demonstrate that mTG can efficiently couple three different acyl-acceptor molecules, l-lysine, glycine ethyl ester, and hydroxylamine, to gliadin peptides and protein. While all three acyl-acceptor molecules were cross-linked to the same Q-residues, not all modifications were equally effective in silencing T-cell reactivity. Finally, we observed that tTG can partially reverse the mTG-catalyzed transamidation by its isopeptidase activity. These results set the stage to determine the impact of these modifications on the baking quality of gluten proteins and in vivo immunogenicity of such food products.

Crystal structure of D-stereospecific amidohydrolase from Streptomyces sp. 82F2 - insight into the structural factors for substrate specificity.

UNLABELLED: D-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2, which catalyzes amide bond formation from d-aminoacyl esters and l-amino acids (aminolysis), can be used to synthesize short peptides with a dl-configuration. We found that DAH can use 1,8-diaminooctane and other amino compounds as acyl acceptors in the aminolysis reaction. Low concentrations of 1,8-diaminooctane inhibited acyl-DAH intermediate formation. By contrast, excess 1,8-diaminooctane promoted aminolysis by DAH, producing d-Phe-1,8-diaminooctane via nucleophilic attack of the diamine on enzyme-bound d-Phe. To clarify the mechanism of substrate specificity and amide bond formation by DAH, the crystal structure of the enzyme that binds 1,8-diaminooctane was determined at a resolution of 1.49 Å. Comparison of the DAH crystal structure with those of other members of the S12 peptidase family indicated that the substrate specificity of DAH arises from its active site structure. The 1,8-diaminooctane molecule binds at the entrance of the active site pocket. The electrkon density map showed that another potential 1,8-diaminooctane binding site, probably with lower affinity, is present close to the active site. The enzyme kinetics and structural comparisons suggest that the location of enzyme-bound diamine can explain the inhibition of the acyl-enzyme intermediate formation, although the bound diamine is too far from the active site for aminolysis. Despite difficulty in locating the diamine binding site for aminolysis definitively, we propose that the excess diamine also binds at or near the second binding site to attack the acyl-enzyme intermediate during aminolysis. DATABASE: The coordinates and structure factors for d-stereospecific amidohydrolase have been deposited in the Protein Data Bank at the Research Collaboratory for Structural Bioinformatics under code: 3WWX.

Biodiesel synthesis by direct transesterification of microalga Botryococcus braunii with continuous methanol reflux.

Direct transesterification of Botryococcus braunii with continuous acyl acceptor reflux was evaluated. This method combines in one step lipid extraction and esterification/transesterification. Fatty acid methyl esters (FAME) synthesis by direct conversion of microalgal biomass was carried out using sulfuric acid as catalyst and methanol as acyl acceptor. In this system, once lipids are extracted, they are contacted with the catalyst and methanol reaching 82%wt of FAME yield. To optimize the reaction conditions, a factorial design using surface response methodology was applied. The effects of catalyst concentration and co-solvent concentration were studied. Hexane was used as co-solvent for increasing lipid extraction performance. The incorporation of hexane in the reaction provoked an increase in FAME yield from 82% (pure methanol) to 95% when a 47%v/v of hexane was incorporated in the reaction. However, the selectivity towards non-saponifiable lipids such as sterols was increased, negatively affecting biodiesel quality.