Crystal structure of dTDP-4-keto-6-deoxy-D-hexulose 3,5-epimerase from Methanobacterium thermoautotrophicum complexed with dTDP

Deoxythymidine diphosphate (dTDP)-4-keto-6-deoxy-d-hexulose 3, 5-epimerase (RmlC) is involved in the biosynthesis of dTDP-l-rhamnose, which is an essential component of the bacterial cell wall. The crystal structure of RmlC from Methanobacterium thermoautotrophicum was determined in the presence and absence of dTDP, a substrate analogue. RmlC is a homodimer comprising a central jelly roll motif, which extends in two directions into longer beta-sheets. Binding of dTDP is stabilized by ionic interactions to the phosphate group and by a combination of ionic and hydrophobic interactions with the base. The active site, which is located in the center of the jelly roll, is formed by residues that are conserved in all known RmlC sequence homologues. The conservation of the active site residues suggests that the mechanism of action is also conserved and that the RmlC structure may be useful in guiding the design of antibacterial drugs.     

Structure of Arabidopsis dehydroquinate dehydratase-shikimate dehydrogenase and implications for metabolic channeling in the shikimate pathway

The bifunctional enzyme dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) catalyzes the dehydration of dehydroquinate to dehydroshikimate and the reduction of dehydroshikimate to shikimate in the shikimate pathway. We report the first crystal structure of Arabidopsis DHQ-SDH with shikimate bound at the SDH site and tartrate at the DHQ site. The interactions observed in the DHQ-tartrate complex reveal a conserved mode for substrate binding between the plant and microbial DHQ dehydratase family of enzymes. The SDH-shikimate complex provides the first direct evidence of the role of active site residues in the catalytic mechanism. Site-directed mutagenesis and mechanistic analysis revealed that Asp 423 and Lys 385 are key catalytic groups and Ser 336 is a key binding group. The arrangement of the two functional domains reveals that the control of metabolic flux through the shikimate pathway is achieved by increasing the effective concentration of dehydroshikimate through the proximity of the two sites.     

Insights into the function of RifI2: Structural and biochemical investigation of a new shikimate dehydrogenase family protein

The shikimate dehydrogenase (SDH) family consists of enzymes with diverse roles in secondary metabolism. The two most widespread members of the family, AroE and YdiB, function in amino acid biosynthesis and quinate catabolism, respectively. Here, we have determined the crystal structure of an SDH homolog belonging to the RifI class, a group of enzymes with proposed roles in antibiotic biosynthesis. The structure of RifI2 from Pseudomonas putida exhibits a number of distinctive features, including a substantial C-terminal truncation and an atypical mode of oligomerization. The active site of the enzyme contains substrate- and cofactor-binding motifs that are significantly different from those of any previously characterized member of the SDH family. These features are reflected in the novel kinetic properties of the enzyme. RifI2 exhibits much lower activity using shikimate as a substrate than AroE, and a strong preference for NAD⁺ instead of NADP⁺ as a cofactor. Moreover, the enzyme has only trace activity using quinate, unlike YdiB. Cocrystallization of RifI2 with NAD⁺ provided the opportunity to determine the mode of cofactor selectivity employed by the enzyme. We complemented this analysis by probing the role of a strictly conserved residue in the cofactor-binding domain, Asn193, by site directed mutagenesis. This study presents the first crystal structure and formal kinetic characterization of a new NAD⁺-dependent member of the SDH family.     

Structural and biochemical investigation of two Arabidopsis shikimate kinases: the heat-inducible isoform is thermostable

The expression of plant shikimate kinase (SK; EC 2.7.1.71), an intermediate step in the shikimate pathway to aromatic amino acid biosynthesis, is induced under specific conditions of environmental stress and developmental requirements in an isoform-specific manner. Despite their important physiological role, experimental structures of plant SKs have not been determined and the biochemical nature of plant SK regulation is unknown. The Arabidopsis thaliana genome encodes two SKs, AtSK1 and AtSK2. We demonstrate that AtSK2 is highly unstable and becomes inactivated at 37 °C whereas the heat-induced isoform, AtSK1, is thermostable and fully active under identical conditions at this temperature. We determined the crystal structure of AtSK2, the first SK structure from the plant kingdom, and conducted biophysical characterizations of both AtSK1 and AtSK2 towards understanding this mechanism of thermal regulation. The crystal structure of AtSK2 is generally conserved with bacterial SKs with the addition of a putative regulatory phosphorylation motif forming part of the adenosine triphosphate binding site. The heat-induced isoform, AtSK1, forms a homodimer in solution, the formation of which facilitates its relative thermostability compared to AtSK2. In silico analyses identified AtSK1 site variants that may contribute to AtSK1 stability. Our findings suggest that AtSK1 performs a unique function under heat stress conditions where AtSK2 could become inactivated. We discuss these findings in the context of regulating metabolic flux to competing downstream pathways through SK-mediated control of steady state concentrations of shikimate.     

Insights into ligand binding and catalysis of a central step in NAD+ synthesis: structures of Methanobacterium thermoautotrophicum NMN adenylyltransferase complexes

Nicotinamide mononucleotide adenylyltransferase (NMNATase) catalyzes the linking of NMN(+) or NaMN(+) with ATP, which in all organisms is one of the common step in the synthesis of the ubiquitous coenzyme NAD(+), via both de novo and salvage biosynthetic pathways. The structure of Methanobacterium thermoautotrophicum NMNATase determined using multiwavelength anomalous dispersion phasing revealed a nucleotide-binding fold common to nucleotidyltransferase proteins. An NAD(+) molecule and a sulfate ion were bound in the active site allowing the identification of residues involved in product binding. In addition, the role of the conserved (16)HXGH(19) active site motif in catalysis was probed by mutagenic, enzymatic and crystallographic techniques, including the characterization of an NMN(+)/SO4(2-) complex of mutant H19A NMNATase.     

The crystal structure of a novel SAM-dependent methyltransferase PH1915 from Pyrococcus horikoshii

The S-adenosyl-L-methionine (SAM)-dependent methyltransferases represent a diverse and biologically important class of enzymes. These enzymes utilize the ubiquitous methyl donor SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. Here we present the crystal structure of PH1915 from Pyrococcus horikoshii OT3, a predicted SAM-dependent methyltransferase. This protein belongs to the Cluster of Orthologous Group 1092, and the presented crystal structure is the first representative structure of this protein family. Based on sequence and 3D structure analysis, we have made valuable functional insights that will facilitate further studies for characterizing this group of proteins. Specifically, we propose that PH1915 and its orthologs are rRNA- or tRNA-specific methyltransferases.     

Synthesis and spectroscopic and X-ray structural characterisation of some para-substituted triaryltin(pentacarbonyl)manganese(I) complexes

A series of para-substituted triaryltin(pentacarbonyl)manganese(I) compounds [(p-XC₆H₄)₃SnMn(CO)₅: II, X=CH₃; III, X=CH₃O; IV, X=CH₃S; V, X=F; VI, X=Cl; VII, X=CH₃S(O₂)] is reported for comparison with the known phenyl analogue I. IR data [ν(CO)] as well as complete ¹¹⁹Sn/⁵⁵Mn/¹³C solution NMR results are given for I-VII. Chemical shifts, ¹¹⁹Sn versus ⁵⁵Mn, except I, correlate well, but have differing single parameter (SP) correlations, ¹¹⁹Sn versus σ_I and ⁵⁵Mn versus σ°_p. These results are compared with previous SP studies of the ¹¹⁹Sn solution NMR spectra of the series, (p-XC₆H₄)₄Sn and (p-XC₆H₄)₃SnY (Y=Cl, Br, I). Full crystal structures are reported for compounds II-VI. All are similar to that of I, with the Mn(CO)₅ moiety being a distorted tetragonal pyramid, and having a quasi-mirror plane through the central C₄MnSnC₃ skeleton. The Ar₃Sn are distorted trigonal propellers with ring torsion angles in the range 30-80°, the exception being IV with one torsion angle of 22°.     

The crystal structure of MT0146/CbiT suggests that the putative precorrin-8w decarboxylase is a methyltransferase

The CbiT and CbiE enzymes participate in the biosynthesis of vitamin B12. They are fused together in some organisms to form a protein called CobL, which catalyzes two methylations and one decarboxylation on a precorrin intermediate. Because CbiE has sequence homology to canonical precorrin methyltransferases, CbiT was hypothesized to catalyze the decarboxylation. We herein present the crystal structure of MT0146, the CbiT homolog from Methanobacterium thermoautotrophicum. The protein shows structural similarity to Rossmann-like S-adenosyl-methionine-dependent methyltransferases, and our 1.9 A cocrystal structure shows that it binds S-adenosyl-methionine in standard geometry near a binding pocket that could accommodate a precorrin substrate. Therefore, MT0146/CbiT probably functions as a precorrin methyltransferase and represents the first enzyme identified with this activity that does not have the canonical precorrin methyltransferase fold.     

Structural and biochemical approaches uncover multiple evolutionary trajectories of plant quinate dehydrogenases

Quinate is produced and used by many plants in the biosynthesis of chlorogenic acids (CGAs). Chlorogenic acids are astringent and serve to deter herbivory. They also function as antifungal agents and have potent antioxidant properties. Quinate is produced at a branch point of shikimate biosynthesis by the enzyme quinate dehydrogenase (QDH). However, little information exists on the identity and biochemical properties of plant QDHs. In this study, we utilized structural and bioinformatics approaches to establish a QDH‐specific primary sequence motif. Using this motif, we identified QDHs from diverse plants and confirmed their activity by recombinant protein production and kinetic assays. Through a detailed phylogenetic analysis, we show that plant QDHs arose directly from bifunctional dehydroquinate dehydratase–shikimate dehydrogenases (DHQD‐SDHs) through different convergent evolutionary events, illustrated by our findings that eudicot and conifer QDHs arose early in vascular plant evolution whereas Brassicaceae QDHs emerged later. This process of recurrent evolution of QDH is further demonstrated by the fact that this family of proteins independently evolved NAD⁺ and NADP⁺ specificity in eudicots. The acquisition of QDH activity by these proteins was accompanied by the inactivation or functional evolution of the DHQD domain, as verified by enzyme activity assays and as reflected in the loss of key DHQD active site residues. The implications of QDH activity and evolution are discussed in terms of plant growth and development.