Shanteri Singh

Solution structure of a late embryogenesis abundant protein (LEA14) from Arabidopsis thaliana, a cellular stress‐related protein

We report the three‐dimensional structure of a late embryogenesis abundant (LEA) protein from Arabidopsis thaliana gene At1g01470.1. This protein is a member of Pfam cluster PF03168, and has been classified as a LEA14 protein. LEA proteins are expressed under conditions of cellular stress, such as desiccation, cold, osmotic stress, and heat. The structure, which was determined by NMR spectroscopy, revealed that the At1g01470.1 protein has an αβ‐fold consisting of one α‐helix and seven β‐strands that form two antiparallel β‐sheets. The closest structural homologs were discovered to be fibronectin Type III domains, which have <7% sequence identity. Because fibronectins from animal cells have been shown to be involved in cell adhesion, cell motility, wound healing, and maintenance of cell shape, it is interesting to note that in plants wounding or stress results in the overexpression of a protein with fibronectin Type III structural features.

The structural biology of enzymes involved in natural product glycosylation

The glycosylation of microbial natural products often dramatically influences the biological and/or pharmacological activities of the parental metabolite. Over the past decade, crystal structures of several enzymes involved in the biosynthesis and attachment of novel sugars found appended to natural products have emerged. In many cases, these studies have paved the way to a better understanding of the corresponding enzyme mechanism of action and have served as a starting point for engineering variant enzymes to facilitate to production of differentially-glycosylated natural products. This review specifically summarizes the structural studies of bacterial enzymes involved in biosynthesis of novel sugar nucleotides.

Comparison of Cell-Based and Cell-Free Protocols for Producing Target Proteins from the Arabidopsis thaliana Genome for Structural Studies

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell‐based and cell‐free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N‐labeled proteins to be analyzed by 1H‐15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell‐based and two involved wheat germ cell‐free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H‐15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC± (protein partially disordered or not in a single stable conformational state), HSQC− (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X− for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell‐free platform offers the advantage of greater genome coverage for NMR‐based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X‐ray structure determination. Proteins 2005.

Glycosyltransferase structural biology and its role in the design of catalysts for glycosylation

Glycosyltransferases (GTs) are ubiquitous in nature and are required for the transfer of sugars to a variety of important biomolecules. This essential enzyme family has been a focus of attention from both the perspective of a potential drug target and a catalyst for the development of vaccines, biopharmaceuticals and small molecule therapeutics. This review attempts to consolidate the emerging lessons from Leloir (nucleotide-dependent) GT structural biology studies and recent applications of these fundamentals toward rational engineering of glycosylation catalysts.

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in kcat/Km of >400-fold and >15-fold for formation of NDP–glucoses and UDP–sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP–sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.

Structure and Mechanism of the Rebeccamycin Sugar 4′-O-Methyltransferase RebM

The 2.65-Å crystal structure of the rebeccamycin 4′-O-methyltransferase RebM in complex with S-adenosyl-l-homocysteine revealed RebM to adopt a typical S-adenosylmethionine-binding fold of small molecule O-methyltransferases (O-MTases) and display a weak dimerization domain unique to MTases. Using this structure as a basis, the RebM substrate binding model implicated a predominance of nonspecific hydrophobic interactions consistent with the reported ability of RebM to methylate a wide range of indolocarbazole surrogates. This model also illuminated the three putative RebM catalytic residues (His140/141 and Asp166) subsequently found to be highly conserved among sequence-related natural product O-MTases from GC-rich bacteria. Interrogation of these residues via site-directed mutagenesis in RebM demonstrated His140 and Asp166 to be most important for catalysis. This study reveals RebM to be a member of the general acid/base-dependent O-MTases and, as the first crystal structure for a sugar O-MTase, may also present a template toward the future engineering of natural product MTases for combinatorial applications.

Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering.

Structural characterization of CalO2: A putative orsellinic acid P450 oxidase in the calicheamicin biosynthetic pathway

Although bacterial iterative Type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products remains poorly understood. Toward this goal, we report the X‐ray structure determination at 2.5 Å resolution and preliminary characterization of the putative orsellenic acid P450 oxidase (CalO2) involved in calicheamicin biosynthesis. These studies represent the first crystal structure for a P450 involved in modifying a bacterial iterative Type I polyketide product and suggest the CalO2‐catalyzed step may occur after CalO3‐catalyzed iodination and may also require a coenzyme A‐ (CoA) or acyl carrier protein‐ (ACP) bound substrate. Docking studies also reveal a putative docking site within CalO2 for the CLM orsellinic acid synthase (CalO5) ACP domain which involves a well‐ordered helix along the CalO2 active site cavity that is unique compared with other P450 structures. Proteins 2009.

Three-dimensional structure of the AAH26994.1 protein from Mus musculus, a putative eukaryotic Urm1.

We have used NMR spectroscopy to determine the solution structure of protein AAH26994.1 from Mus musculus and propose that it represents the first three‐dimensional structure of a ubiquitin‐related modifier 1 (Urm1) protein. Amino acid sequence comparisons indicate that AAH26994.1 belongs to the Urm1 family of ubiquitin‐like modifier proteins. The best characterized member of this family has been shown to be involved in nutrient sensing, invasive growth, and budding in yeast. Proteins in this family have only a weak sequence similarity to ubiquitin, and the structure of AAH26994.1 showed a much closer resemblance to MoaD subunits of molybdopterin synthases (known structures are of three bacterial MoaD proteins with 14%–26% sequence identity to AAH26994.1). The structures of AAH26994.1 and the MoaD proteins each contain the signature ubiquitin secondary structure fold, but all differ from ubiquitin largely in regions outside of this fold. This structural similarity bolsters the hypothesis that ubiquitin and ubiquitin‐related proteins evolved from a protein‐based sulfide donor system of the molybdopterin synthase type.

Functionalized anodic aluminum oxide membrane-electrode system for enzyme immobilization.

A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions—a one-step “reverse” sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6–20 min–1 on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min–1. UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process.