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Abstract: 2′-Deoxycytidylate deaminase [or deoxycytidine-5′-monophosphate (dCMP) deaminase, dCD] catalyzes the deamination of dCMP to deoxyuridine-5′-monophosphate to provide the main nucleotide substrate for thymidylate synthase, which is important in DNA synthesis. The activity of this homohexameric enzyme is allosterically regulated by deoxycytidine-5′-triphosphate (dCTP) as an activator and by deoxythymidine-5′-triphosphate as an inhibitor. In this article, we report the crystal structures of dCMP deaminase from Streptococcus mutans and its complex with dCTP and an intermediate analog at resolutions of 3.0 and 1.66 Å. The protein forms a hexamer composed of subunits adopting a three-layer α/β/α sandwich fold. The positive allosteric regulator dCTP mainly binds at the interface between two monomers in a molar ratio of 1:1 and rearranges the neighboring interaction networks. Structural comparisons and sequence alignments revealed that dCMP deaminase from Streptococcus mutans belongs to the cytidine deaminase superfamily, wherein the proteins exhibit a similar catalytic mechanism. In addition to the two conserved motifs involved in the binding of Zn2+, a new conserved motif, (G43YNG46), related to the binding of dCTP was also identified. N-terminal Arg4, a key residue located between two monomers, binds strongly to the γ phosphate group of dCTP. The regulation signal was transmitted by Arg4 from the allosteric site to the active site via modifications in the interactions at the interface where the substrate-binding pocket was involved and the relocations of Arg26, His65, Tyr120, and Arg121 to envelope the active site in order to stabilize substrate binding in the complex. Based on the enzyme–regulator complex structure observed in this study, we propose an allosteric mechanism for dCD regulation. [Copyright &y& Elsevier]

Abstract: Cobalamin-independent methionine synthase (MetE) catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to l-homocysteine to form methionine. Previous studies have shown that the MetE active site coordinates a zinc atom, which is thought to act as a Lewis acid and plays a role in the activation of thiol. Extended X-ray absorption fine structure studies and mutagenesis experiments identified the zinc-binding site in MetE from Escherichia coli. Further structural investigations of MetE from Thermotoga maritima lead to the proposition of two models: “induced fit” and “dynamic equilibrium”, to account for the catalytic mechanisms of MetE. Here, we present crystal structures of oxidized and zinc-replete MetE from Streptococcus mutans at the physiological pH. The structures reveal that zinc is mobile in the active center and has the possibility to invert even in the absence of homocysteine. These structures provide evidence for the dynamic equilibrium model. [Copyright &y& Elsevier]

Abstract: Glucosamine 6-phosphate deaminase (NagB) catalyzes the conversion of d-glucosamine 6-phosphate (GlcN6P) to d-fructose 6-phosphate and ammonia. This reaction is the final step of N-acetylglucosamine utilization and decides its metabolic fate. The enzyme from Streptococcus mutans belongs to the monomeric subfamily of NagB. The crystal structure of the native SmuNagB (NagB from S. mutans) presented here, compared with the structures of its homologs BsuNagB (NagB from Bacillus subtilis) and EcoNagB (NagB from E. coli), implies a conformational change of the ‘lid’ motif in the activation of the monomeric NagB enzyme. We have also captured the enzyme–substrate intermediate complex of the NagB family at low pH, where a remarkable loss of the catalytic activity of SmuNagB was detected. The enzyme–substrate intermediate presents the initial step of the GlcN6P deaminase reaction. The structural evidence (1) supports the α-anomer of GlcN6P as the specific natural substrate of NagB; (2) displays the substrate-binding pocket at the active site; and (3) together with the site-directed mutagenesis studies, demonstrates the ring-opening mechanism of an Asn-His-Glu triad that performs the proton transfer from O1 to O5 to open the sugar ring. [Copyright &y& Elsevier]

Abstract: C4-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C4-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB–DctD system is the first identified TCS for C4-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C4 succinate, and a complex with C3 malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per–Arnt–Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C4-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases. [Copyright &y& Elsevier]