An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in L-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional β-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an L-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature

Evolution of enzymatic activities in the enolase superfamily: L-fuconate dehydratase from Xanthomonas campestris

Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report we use both genome (operon) context and screening of a library of acid sugars to assign the l-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI:21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups:  three carboxylate ligands for the essential Mg2+ located at the ends of the third, fourth, and fifth β-strands in the (β/α)7β-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second β-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and sixth β-strands (His 351 and Asp 324, respectively), and a Glu at the end of the eighth β-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous β-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting enol, likely by the conjugate acid of Lys 220, to yield the 2-keto-3-deoxy-l-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity:  In addition to l-fuconate, FucD also catalyzes the dehydration of l-galactonate, d-arabinonate, d-altronate, l-talonate, and d-ribonate. The dehydrations of l-fuconate, l-galactonate, and d-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of l-talonate and d-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields l-galactonate and d-arabinonate in competition with dehydration. The functional promiscuity discovered for FucD highlights possible structural mechanisms for evolution of function in the enolase superfamil

Evolution of Enzymatic Activities in the Enolase Superfamily: d-Mannonate Dehydratase from Novosphingobium aromaticivorans

The D-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal α+β capping domain and a (β/α)7β-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth β-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second β-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth β-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second β-strand and Arg 147 at the end of the second β-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third β-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the only conserved residues in the enolase superfamily, establishing the primary functional importance of the Mg2+-assisted strategy for stabilizing the enolate anion intermediate

Magnetic Hysteresis at 10 K in Single Molecule Magnet Self‐Assembled on Gold

Tremendous progress in the development of single molecule magnets (SMMs) raises the question of their device integration. On this route, understanding the properties of low‐dimensional assemblies of SMMs, in particular in contact with electrodes, is a necessary but difficult step. Here, it is shown that fullerene SMM self‐assembled on metal substrate from solution retains magnetic hysteresis up to 10 K. Fullerene‐SMM DySc2N@C80 and Dy2ScN@C80 are derivatized to introduce a thioacetate group, which is used to graft SMMs on gold. Magnetic properties of grafted SMMs are studied by X‐ray magnetic circular dichroism and compared to the films of nonderivatized fullerenes prepared by sublimation. In self‐assembled films, the magnetic moments of the Dy ions are preferentially aligned parallel to the surface, which is different from the disordered orientation of endohedral clusters in nonfunctionalized fullerenes. Whereas chemical derivatization reduces the blocking temperature of magnetization and narrows the hysteresis of Dy2ScN@C80, for DySc2N@C80 equally broad hysteresis is observed as in the fullerene multilayer. Magnetic bistability in the DySc2N@C80 grafted on gold is sustained up to 10 K. This study demonstrates that self‐assembly of fullerene‐SMM derivatives offers a facile solution‐based procedure for the preparation of functional magnetic sub‐monolayers with excellent SMM performance