A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

Mechanism and Structure of γ-Resorcylate Decarboxylase.

γ-Resorcylate decarboxylase (γ-RSD) has evolved to catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate to resorcinol in a nonoxidative fashion. This enzyme is of significant interest because of its potential for the production of γ-resorcylate and other benzoic acid derivatives under environmentally sustainable conditions. Kinetic constants for the decarboxylation of 2,6-dihydroxybenzoate catalyzed by γ-RSD from Polaromonas sp. JS666 are reported, and the enzyme is shown to be active with 2,3-dihydroxybenzoate, 2,4,6-trihydroxybenzoate, and 2,6-dihydroxy-4-methylbenzoate. The three-dimensional structure of γ-RSD with the inhibitor 2-nitroresorcinol (2-NR) bound in the active site is reported. 2-NR is directly ligated to a Mn2+ bound in the active site, and the nitro substituent of the inhibitor is tilted significantly from the plane of the phenyl ring. The inhibitor exhibits a binding mode different from that of the substrate bound in the previously determined structure of γ-RSD from Rhizobium sp. MTP-10005. On the basis of the crystal structure of the enzyme from Polaromonas sp. JS666, complementary density functional calculations were performed to investigate the reaction mechanism. In the proposed reaction mechanism, γ-RSD binds 2,6-dihydroxybenzoate by direct coordination of the active site manganese ion to the carboxylate anion of the substrate and one of the adjacent phenolic oxygens. The enzyme subsequently catalyzes the transfer of a proton to C1 of γ-resorcylate prior to the actual decarboxylation step. The reaction mechanism proposed previously, based on the structure of γ-RSD from Rhizobium sp. MTP-10005, is shown to be associated with high energies and thus less likely to be correct.

Purification, crystallization and structural elucidation of D-galactaro-1,4-lactone cycloisomerase from Agrobacterium tumefaciens involved in pectin degradation.

Pectin is found in the cell wall of plants and is often discarded as waste. A number of research groups are interested in redirecting this biomass waste stream for the production of fuel and bulk chemicals. The primary monomeric subunit of this polysaccharide is D-galacturonate, a six-carbon acid sugar that is degraded in a five-step pathway to central metabolic intermediates by some bacteria, including Agrobacterium tumefaciens. In the third step of the pathway, D-galactaro-1,4-lactone is converted to 2-keto-3-deoxy-L-threo-hexarate by a member of the mandelate racemase subgroup of the enolase superfamily with a novel activity for the superfamily. The 1.6 Šresolution structure of this enzyme was determined, revealing an overall modified (ß/α)7ß TIM-barrel domain, a hallmark of the superfamily. D-Galactaro-1,4-lactone was manually docked into the active site located at the interface between the N-terminal lid domain and the C-terminal barrel domain. On the basis of the position of the lactone in the active site, Lys166 is predicted to be the active-site base responsible for abstraction of the α proton. His296 on the opposite side of the active site is predicted to be the general acid that donates a proton to the ß carbon as the lactone ring opens. The lactone ring appears to be oriented within the active site by stacking interactions with Trp298.

Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification.

Enzyme-mediated modifications at the wobble position of tRNAs are essential for the translation of the genetic code. We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs. CmoB is distinctive in that it is the only known member of the SAM-dependent methyltransferase (SDMT) superfamily that utilizes a naturally occurring SAM analog as the alkyl donor to fulfill a biologically meaningful function. Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM. Complementary high-resolution structures of the apo- and Cx-SAM bound CmoB reveal the determinants responsible for this remarkable discrimination. Together, these studies provide mechanistic insight into the enzymatic and non-enzymatic feature of this alkyl transfer reaction which affords the broadened specificity required for tRNAs to recognize multiple synonymous codons.

Divergent evolution in enolase superfamily: strategies for assigning functions.

Nature's strategies for evolving catalytic functions can be deciphered from the information contained in the rapidly expanding protein sequence databases. However, the functions of many proteins in the protein sequence and structure databases are either uncertain (too divergent to assign function based on homology) or unknown (no homologs), thereby limiting the utility of the databases. The mechanistically diverse enolase superfamily is a paradigm for understanding the structural bases for evolution of enzymatic function. We describe strategies for assigning functions to members of the enolase superfamily that should be applicable to other superfamilies.

Structure and dynamics of the actin filament.

The structures of filamentous Mg-ATP-actin (F actin) in the presence and absence of KCl have been mapped with hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry (MS) analysis revealed 52 reactive side-chain sites from 27 distinct peptides within actin. The reactivities of these probe sites with *OH in the F-actin states are compared with those of Mg-ATP-G-actin (monomers) analyzed previously [Guan, J.-Q. et al. (2003) Biochemistry 42, 11992-12000]. Filament-dependent protection within subdomains 2, 3, and 4 and at the C terminus is consistent with longitudinal contacts of monomers within the filament helical structure as predicted by the Holmes model. In the absence of KCl, the extent of filament-dependent protection rarely reached 3-fold, consistent with a highly dynamic filament characterized by relatively weak interactions between actin protomers. However, in the presence of KCl, the extents of protection are significantly increased, consistent with a well-ordered, more tightly packed filament structure. Filament-dependent enhancements of reactivity not predicted by the Holmes model are seen for a peptide that overlaps the "hydrophobic plug" (H-plug) region and for a peptide that forms contacts with the polyphosphate moiety of the bound nucleotide. Overall, these data are both consistent with and complementary to a recent deuterium-exchange MS study of filamentous actin [Chik, J. K., and Schriemer, D.C. (2003) J. Mol. Biol. 334, 373-385], which also did not detect any burial of the H plug upon formation of filaments.