Exploring the Structure-Function Relationships in a 5-Aminolevulinic Acid Synthase and the Use of Protein Engineering to Expand its Substrate Range.

5-Aminolevulinate synthase (ALAS) is a PLP-dependent enzyme that catalyzes the production of 5-aminolevulinate from succinyl-CoA and glycine. Its ability to catalyze the essentially irreversible - bond formation has significant potential in chemoenzymatic synthesis of α-amino ketones. Native ALAS, unfortunately, is extremely substrate-selective, and this seriously limits its synthetic utility.

Optimization of the Recovery of Secondary Metabolites from Defatted Meal and Its Effects on the Extractability and Functional Properties of Proteins.

The sustainable extraction of secondary metabolites from Brassica agro-industrial by-products often involves the use of high concentrations of ethanol, and/or high temperatures, which tends to decrease the efficiency of protein extraction (yield, profile, etc.). To understand the limits of the combination of these two extraction processes, aqueous ethanol extraction of secondary metabolites (e.

Sinapic Acid and Sinapate Esters in : Innate Accumulation, Biosynthesis, Accessibility Chemical Synthesis or Recovery From Biomass, and Biological Activities.

Sinapic acid (SinA) and corresponding esters are secondary metabolites abundantly found in plants of Brassica family. Belonging to the family of -hydroxycinnamic acids, SinA and its esters analogues are present in different plant parts and involved in multiple biological processes . Moreover, these metabolites are also found in relatively large quantities in agro-industrial wastes.

Semi-rational approach to expand the Acyl-CoA Chain length tolerance of Sphingomonas paucimobilis serine palmitoyltransferase.

Serine palmitoyltransferase (SPTase), the first enzyme of the sphingolipid biosynthesis pathway, produces 3-ketodihydrosphingosine by a Claisen-like condensation/decarboxylation reaction of l-Ser and palmitoyl-CoA (n-C-CoA). Previous structural analysis of Sphingomonas paucimobilis SPTase (SpSPTase) revealed a dynamic active site loop (RPPATP; amino acids 378-383) in which R378 (underlined) forms a salt bridge with the carboxylic acid group of the PLP : l-Ser external aldimine. We hypothesized that this interaction might play a key role in acyl group substrate selectivity and therefore performed site-saturation mutagenesis at position 378 based on semi-rational design to expand tolerance for shorter acyl-CoA's.

Application of Acetyl-CoA synthetase from Methanothermobacter thermautotrophicus to non-native substrates.

The substrate selectivity of the TrpGly mutant of Methanothermobacter thermautotrophicus acetyl-CoA synthetase (TrpGly MT-ACS1) was explored. The goal was to identify its substrate range, particularly for functionalized carboxylic acid substrates that would allow post-synthesis functionalization of CoA thioesters or downstream products using metathesis or Click chemistry. Relative activities were determined by in situ formation of acyl-hydroxamate iron (III) complexes.

Effects of long-term exposure to elevated temperature on Zea mays endosperm development during grain fill.

Cereal yields decrease when grain fill proceeds under conditions of prolonged, moderately elevated temperatures. Endosperm-endogenous processes alter both rate and duration of dry weight gain, but underlying mechanisms remain unclear. Heat effects could be mediated by either abnormal, premature cessation of storage compound deposition or accelerated implementation of normal development.

Progress in using threonine aldolases for preparative synthesis.

Three threonine aldolases (TAs) were cloned and overexpressed in Escherichia coli (Aeromonas jandaeil-allo-threonine aldolase, E. colil-threonine aldolase and Thermotoga maritimal-allo-threonine aldolase). A Design of Experiments strategy was used to identify optimal reaction conditions for each enzyme.

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm.

Maize () mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by include and that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants.

Role of Polymer Architecture on the Activity of Polymer-Protein Conjugates for the Treatment of Accelerated Bone Loss Disorders.

Polymers of similar molecular weights and chemical constitution but varying in their macromolecular architectures were conjugated to osteoprotegerin (OPG) to determine the effect of polymer topology on protein activity in vitro and in vivo. OPG is a protein that inhibits bone resorption by preventing the formation of mature osteoclasts from the osteoclast precursor cell. Accelerated bone loss disorders, such as osteoporosis, rheumatoid arthritis, and metastatic bone disease, occur as a result of increased osteoclastogenesis, leading to the severe weakening of the bone.

Applications of protein engineering to members of the old yellow enzyme family.

In the 20 years since Massey's initial report in 1995, interest in using alkene reductases to prepare chiral intermediates for synthesis has grown rapidly. While native alkene reductases often show very high stereoselectivities toward favorable substrates, these enzymes have somewhat size-restricted active sites that limit their substrate ranges to small alkenes. In addition, most alkene reductases have the same stereoselectivities, which makes it difficult to access the "other" product enantiomers.

Enhancing the heat stability and kinetic parameters of the maize endosperm ADP-glucose pyrophosphorylase using iterative saturation mutagenesis.

Iterative saturation mutagenesis (ISM) has been used to improve the thermostability of maize endosperm ADP-glucose pyrophosphorylase (AGPase), a highly-regulated, rate-limiting and temperature-sensitive enzyme essential for starch biosynthesis. The thermo-sensitivity of heterotetrameric AGPase has been linked to grain loss in cereals and improving this property might therefore have direct impacts on grain yield. Nine amino acids were selected for site-saturation mutagenesis on the basis of elevated B-factors in the crystal structure of the closest available homolog (a small subunit homotetramer of potato AGPase).

Pichia stipitis OYE 2.6 variants with improved catalytic efficiencies from site-saturation mutagenesis libraries.

An earlier directed evolution project using alkene reductase OYE 2.6 from Pichia stipitis yielded 13 active site variants with improved properties toward three homologous Baylis-Hillman adducts. Here, we probed the generality of these improvements by testing the wild-type and all 13 variants against a panel of 16 structurally-diverse electron-deficient alkenes.

Residues Controlling Facial Selectivity in an Alkene Reductase and Semirational Alterations to Create Stereocomplementary Variants.

A systematic saturation mutagenesis campaign was carried out on an alkene reductase from (OYE 2.6) to develop variants with reversed stereoselectivities. Wild-type OYE 2.

Influence of Cofactor Regeneration Strategies on Preparative-Scale, Asymmetric Carbonyl Reductions by Engineered .

This study was designed to determine whether whole cells or crude enzyme extracts are more effective for preparative-scale ketone reductions by dehydrogenases as well as learning which cofactor regeneration scheme is most effective. Based on results from three representative ketone substrates (an α-fluoro-β-keto ester, a -trifluoromethylated acetophenone, and a symmetrical β-diketone), our results demonstrate that several nicotinamide cofactor regeneration strategies can be applied to preparative-scale dehydrogenase-catalyzed reactions successfully.

Threonine aldolases.

Threonine aldolases catalyze the pyridoxal phosphate-dependent condensation between small amino acids (principally glycine) and aldehydes such as acetaldehyde. Carbon-carbon bond formation involves forming two adjacent chiral centers. As a rule, threonine aldolases are very stereoselective for α-carbon configuration but show modest selectivity at the β-carbon.

The potato tuber, maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition.

ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA).

Library construction and evaluation for site saturation mutagenesis.

We developed a method for creating and evaluating site-saturation libraries that consistently yields an average of 27.4±3.0 codons of the 32 possible within a pool of 95 transformants.

Deciphering the kinetic mechanisms controlling selected plant ADP-glucose pyrophosphorylases.

ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes.

The structural basis of HLA-associated drug hypersensitivity syndromes.

Recent data suggest alternative mechanisms that promote human leukocyte antigen (HLA)-associated drug syndromes. Hypersensitive responses have been attributed to drug interactions with HLA molecules, peptides presented by HLA molecules and T-cell antigen receptors. Definition of an increasing number of HLA-associated drug syndromes suggests that polymorphism in the antigen-binding cleft residues influence recognition of specific drugs.

A shrunken-2 transgene increases maize yield by acting in maternal tissues to increase the frequency of seed development.

The maize (Zea mays) shrunken-2 (Sh2) gene encodes the large subunit of the rate-limiting starch biosynthetic enzyme, ADP-glucose pyrophosphorylase. Expression of a transgenic form of the enzyme with enhanced heat stability and reduced phosphate inhibition increased maize yield up to 64%. The extent of the yield increase is dependent on temperatures during the first 4 d post pollination, and yield is increased if average daily high temperatures exceed 33 °C.

Drug-delivery strategies by using template-synthesized nanotubes.

Encapsulating drugs within hollow nanotubes offers several advantages, including protection from degradation, the possibility of targeting desired locations, and drug release only under specific conditions. Template synthesis utilizes porous membranes prepared from alumina, polycarbonate, or other materials that can be dissolved under specific conditions. The method allows for great control over the lengths and diameters of nanotubes; moreover, tubes can be constructed from a wide variety of tube materials including proteins, DNA, silica, carbon, and chitosan.

Fabrication of biodegradable nano test tubes by template synthesis.

Aims: Recent publications have suggested that cylindrically shaped drug-delivery carriers have an advantage over carriers based on spherical particles in both blood circulation and cell internalization rates. For this reason, this article introduces a method to fabricate hollow, uniform, biodegradable chitosan nano test tubes for applications in drug delivery.

Methods: A nanoporous alumina template membrane was used to fabricate hollow chitosan nano test tubes.

Towards preparative-scale, biocatalytic alkene reductions.

Simple strategies for using alkene reductase enzymes to produce gram-scale quantities of both (R)- and (S)-citronellal have been developed. The methodology is easily accessible to non-specialist laboratories, allowing alkene reductases to be added to the toolbox of routine synthetic transformations.

Studies of the kinetic mechanism of maize endosperm ADP-glucose pyrophosphorylase uncovered complex regulatory properties.

ADP-glucose pyrophosphorylase catalyzes the synthesis of ADP-glucose (ADP-Glc) from Glc-1-phosphate (G-1-P) and ATP. Kinetic studies were performed to define the nature of the reaction, both in the presence and absence of allosteric effector molecules. When 3-phosphoglycerate (3-PGA), the putative physiological activator, was present at a saturating level, initial velocity studies were consistent with a Theorell-Chance BiBi mechanism and product inhibition data supported sequential binding of ATP and G-1-P, followed by ordered release of pyrophosphate and ADP-Glc.

Probing allosteric binding sites of the maize endosperm ADP-glucose pyrophosphorylase.

Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA).