William Edstrom

Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB

Nucleic acid damage by environmental and endogenous alkylation reagents creates lesions that are both mutagenic and cytotoxic, with the latter effect accounting for their widespread use in clinical cancer chemotherapy. Escherichia coli AlkB and the homologous human proteins ABH2 and ABH3 (refs 5, 7) promiscuously repair DNA and RNA bases damaged by SN2 alkylation reagents, which attach hydrocarbons to endocyclic ring nitrogen atoms (N1 of adenine and guanine and N3 of thymine and cytosine). Although the role of AlkB in DNA repair has long been established based on phenotypic studies, its exact biochemical activity was only elucidated recently after sequence profile analysis revealed it to be a member of the Fe-oxoglutarate-dependent dioxygenase superfamily. These enzymes use an Fe(ii) cofactor and 2-oxoglutarate co-substrate to oxidize organic substrates. AlkB hydroxylates an alkylated nucleotide base to produce an unstable product that releases an aldehyde to regenerate the unmodified base. Here we have determined crystal structures of substrate and product complexes of E. coli AlkB at resolutions from 1.8 to 2.3 Å. Whereas the Fe-2-oxoglutarate dioxygenase core matches that in other superfamily members, a unique subdomain holds a methylated trinucleotide substrate into the active site through contacts to the polynucleotide backbone. Amide hydrogen exchange studies and crystallographic analyses suggest that this substrate-binding ‘lid’ is conformationally flexible, which may enable docking of diverse alkylated nucleotide substrates in optimal catalytic geometry. Different crystal structures show open and closed states of a tunnel putatively gating O2 diffusion into the active site. Exposing crystals of the anaerobic Michaelis complex to air yields slow but substantial oxidation of 2-oxoglutarate that is inefficiently coupled to nucleotide oxidation. These observations suggest that protein dynamics modulate redox chemistry and that a hypothesized migration of the reactive oxy-ferryl ligand on the catalytic Fe ion may be impeded when the protein is constrained in the crystal lattice.

Visually-guided protein crystal manipulation using micromachined silicon tools

We present a system for protein crystal micromanipulation with focus on automated crystal mounting for the purposes of X-ray data collection. The system features a set of newly designed micropositioner end-effectors we call microshovels which address some limitations of the traditional cryogenic loops. We have used micro-electrical mechanical system (MEMS) techniques to design and manufacture various shapes and quantities of microshovels. Visual feedback from a camera mounted on the microscope is used to control the micropositioner as it lowers a microshovel into the liquid containing the crystals and approaches a selected crystal for pickup. We present experimental results that illustrate the applicability of our approach.

Crystal structures of two bacterial 3-hydroxy-3-methylglutaryl-CoA lyases suggest a common catalytic mechanism among a family of TIM barrel metalloenzymes cleaving carbon-carbon bonds.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 Å resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name “DRE-TIM metallolyases” for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis results and can guide subsequent studies directed at definitive experimental elucidation of this enzymes reaction mechanism.

The 2.35 A structure of the TenA homolog from Pyrococcus furiosus supports an enzymatic function in thiamine metabolism.

TenA (transcriptional enhancer A) has been proposed to function as a transcriptional regulator based on observed changes in gene-expression patterns when overexpressed in Bacillus subtilis. However, studies of the distribution of proteins involved in thiamine biosynthesis in different fully sequenced genomes have suggested that TenA may be an enzyme involved in thiamine biosynthesis, with a function related to that of the ThiC protein. The crystal structure of PF1337, the TenA homolog from Pyrococcus furiosus, is presented here. The protomer comprises a bundle of alpha-helices with a similar tertiary structure and topology to that of human heme oxygenase-1, even though there is no significant sequence homology. A solvent-sequestered cavity lined by phylogenetically conserved residues is found at the core of this bundle in PF1337 and this cavity is observed to contain electron density for 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate, the product of the ThiC enzyme. In contrast, the modestly acidic surface of PF1337 shows minimal levels of sequence conservation and a dearth of the basic residues that are typically involved in DNA binding in transcription factors. Without significant conservation of its surface properties, TenA is unlikely to mediate functionally important protein-protein or protein-DNA interactions. Therefore, the crystal structure of PF1337 supports the hypothesis that TenA homologs have an indirect effect in altering gene-expression patterns and function instead as enzymes involved in thiamine metabolism.

Automated streak-seeding with micromachined silicon tools

This report presents a new approach to streak-seeding based on custom-designed silicon microtools. Experimental data show that the microtools produce similar results to the commonly used boar bristles. One advantage to using silicon is that it is rigid and can easily serve as an accurately calibrated end-effector on a micro-robotic system. Additionally, the fabrication technology allows the production of microtools of various shapes and sizes. A working prototype of an automatic streak-seeding system based on these microtools was built and successfully applied for protein crystallization.

Microrobotic Streak Seeding for Protein Crystal Growth

We present a microrobotic system for protein crystal micromanipulation tasks. The focus in this paper is on the task known to crystallographers as streak seeding – it is used to entice certain protein crystals to grow. Our system features a set of custom designed micropositioner end-effectors we call microshovels to replace traditional tools used by crystallographers for this task. We have used micro-electrical mechanical system (MEMS) techniques to design and manufacture various shapes and quantities of microshovels. Visual input from a camera mounted on the microscope is used to detect the locations of the source crystals which the tool needs to touch as well as the locations of the target protein droplets for seeding. We present experimental results that illustrate the applicability of our approach.

A Microrobotic System For Protein Streak Seeding

We present a microrobotic system for protein crystal micromanipulation tasks. The focus in this report is on a task called streak seeding, which is used by crystallographers to entice certain protein crystals to grow. Our system features a set of custom designed micropositioner end-effectors we call microshovels to replace traditional tools used by crystallographers for this task. We have used micro-electrical mechanical system (MEMS) techniques to design and manufacture various shapes and quantities of microshovels. Visual feedback from a camera mounted on the microscope is used to control the micropositioner as it lowers a microshovel into the liquid containing the crystals for poking and streaking. We present experimental results that illustrate the applicability of our approach.