RESUMO
Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug's direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition (mfmA and mfmB) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (kcat/KM) of 9.6 × 103 M-1s-1 and KM for metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.
Assuntos
Diabetes Mellitus Tipo 2 , Guanidina/análogos & derivados , Metformina , Microbiota , Ureia/análogos & derivados , Humanos , Metformina/metabolismo , Águas Residuárias , Níquel , Hidrolases/genética , Preparações FarmacêuticasRESUMO
Enzymes possessing the nickel-pincer nucleotide (NPN) cofactor catalyze C2 racemization or epimerization reactions of α-hydroxyacid substrates. LarB initiates synthesis of the NPN cofactor from nicotinic acid adenine dinucleotide (NaAD) by performing dual reactions: pyridinium ring C5 carboxylation and phosphoanhydride hydrolysis. Here, we show that LarB uses carbon dioxide, not bicarbonate, as the substrate for carboxylation and activates water for hydrolytic attack on the AMP-associated phosphate of C5-carboxylated-NaAD. Structural investigations show that LarB has an N-terminal domain of unique fold and a C-terminal domain homologous to aminoimidazole ribonucleotide carboxylase/mutase (PurE). Like PurE, LarB is octameric with four active sites located at subunit interfaces. The complex of LarB with NAD+, an analog of NaAD, reveals the formation of a covalent adduct between the active site Cys221 and C4 of NAD+, resulting in a boat-shaped dearomatized pyridine ring. The formation of such an intermediate with NaAD would enhance the reactivity of C5 to facilitate carboxylation. Glu180 is well positioned to abstract the C5 proton, restoring aromaticity as Cys221 is expelled. The structure of as-isolated LarB and its complexes with NAD+ and the product AMP identify additional residues potentially important for substrate binding and catalysis. In combination with these findings, the results from structure-guided mutagenesis studies lead us to propose enzymatic mechanisms for both the carboxylation and hydrolysis reactions of LarB that are distinct from that of PurE.
Assuntos
Cisteína/química , Hidrolases/metabolismo , Lactobacillus plantarum/enzimologia , Níquel/metabolismo , Nucleotídeos/biossíntese , Piridinas/química , Racemases e Epimerases/metabolismo , Carboxiliases , Catálise , Cristalografia por Raios X , Hidrolases/química , Hidrólise , Modelos Moleculares , Conformação Proteica , Racemases e Epimerases/química , Especificidade por SubstratoRESUMO
LarB catalyzes the first step of biosynthesis for the nickel-pincer nucleotide cofactor by converting nicotinic acid adenine dinucleotide (NaAD) to AMP and pyridinium-3,5-biscarboxylic acid mononucleotide (P2CMN). Prior studies had shown that LarB uses CO2 for substrate carboxylation and reported the structure of a Lactiplantibacillus plantarum LarB·NAD+ complex, revealing a covalent linkage between Cys221 and C4 of the pyridine ring. This interaction was proposed to promote C5 carboxylation, with C5-carboxylated-NaAD suggested to activate magnesium-bound water, leading to phosphoanhydride hydrolysis. Here, we extended the analysis of wild-type LarB by using ultraviolet-visible spectroscopy to obtain additional evidence for cysteinyl side chain attachment to the ring of NAD+, thus demonstrating that this linkage is not a crystallization artifact. Using the S127A variant of L. plantarum LarB, a form of the enzyme with a reduced rate of NaAD hydrolysis, we examined its interaction with the authentic substrate. The intermediate arising from C5 carboxylation of NaAD, dinicotinic acid adenine dinucleotide (DaAD), was identified by using mass spectrometry. S127A LarB exhibited spectroscopic evidence of a Cys221-NAD+ adduct, but a covalent enzyme-NaAD linkage was not detectable. We determined the S127A LarB·NaAD structure, providing new insights into the enzyme mechanism, and tentatively identified the position and mode of CO2 binding. The crystal structure revealed the location of the side chain for Glu180, which was previously disordered, but showed that it is not well positioned to abstract the C5 proton in the adduct species to restore aromaticity as Cys221 is expelled. Based on these combined results, we propose a revised catalytic mechanism of LarB..
Assuntos
NAD , Níquel , NAD/metabolismo , Níquel/química , Dióxido de Carbono , Nucleotídeos/metabolismo , Catálise , Cristalografia por Raios XRESUMO
The non-heme Fe(II) and 2-oxoglutarate (2OG) dependent ethylene-forming enzyme (EFE) catalyzes both ethylene generation and L-Arg hydroxylation. Despite experimental and computational progress in understanding the mechanism of EFE, no EFE variant has been optimized for ethylene production while simultaneously reducing the L-Arg hydroxylation activity. In this study, we show that the two L-Arg binding conformations, associated with different reactivity preferences in EFE, lead to differences in the intrinsic electric field (IntEF) of EFE. Importantly, we suggest that applying an external electric field (ExtEF) along the Fe-O bond in the EFE·Fe(III)·OO-Ë·2OG·L-Arg complex can switch the EFE reactivity between L-Arg hydroxylation and ethylene generation. Furthermore, we explored how applying an ExtEF alters the geometry, electronic structure of the key reaction intermediates, and the individual energy contributions of second coordination sphere (SCS) residues through combined quantum mechanics/molecular mechanics (QM/MM) calculations. Experimentally generated variant forms of EFE with alanine substituted for SCS residues responsible for stabilizing the key intermediates in the two reactions of EFE led to changes in enzyme activity, thus demonstrating the key role of these residues. Overall, the results of applying an ExtEF indicate that making the IntEF of EFE less negative and stabilizing the off-line binding of 2OG is predicted to increase ethylene generation while reducing L-Arg hydroxylation.
Assuntos
Arginina , Compostos Férricos , Hidroxilação , Arginina/química , Etilenos/químicaRESUMO
The nickel-pincer nucleotide (NPN) coenzyme, a substituted pyridinium mononucleotide that tri-coordinates nickel, was first identified covalently attached to a lysine residue in the LarA protein of lactate racemase. Starting from nicotinic acid adenine dinucleotide, LarB carboxylates C5 of the pyridinium ring and hydrolyzes the phosphoanhydride, LarE converts the C3 and C5 carboxylates to thiocarboxylates, and LarC incorporates nickel to form a C-Ni and two S-Ni bonds, during the biosynthesis of this cofactor. LarB uses a novel carboxylation mechanism involving the transient formation of a cysteinyl-pyridinium adduct. Depending on the source of the enzyme, LarEs either catalyze a sacrificial sulfur transfer from a cysteinyl side chain resulting in the formation of dehydroalanine or they utilize a [4Fe-4S] cluster bound by three cysteine residues to accept and transfer a non-core sulfide atom. LarC is a CTP-dependent enzyme that cytidinylylates its substrate, adds nickel, then hydrolyzes the product to release NPN and CMP. Homologs of the four lar genes are widely distributed in microorganisms, with some species containing multiple copies of larA whereas others lack this gene, consistent with the cofactor serving other functions. Several LarA-like proteins were shown to catalyze racemase or epimerase activities using 2-hydroxyacid substrates other than lactic acid. Thus, lactate racemase is the founding member of a large family of NPN-containing enzymes.
Assuntos
Lactobacillus plantarum , Níquel , Coenzimas/química , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Níquel/química , Níquel/metabolismo , Nucleotídeos/metabolismo , Enxofre/metabolismoRESUMO
LarE from Lactobacillus plantarum is an ATP-dependent sulfur transferase that sacrifices its Cys176 sulfur atom to form a dehydroalanine (Dha) side chain during biosynthesis of the covalently linked nickel-pincer nucleotide (NPN) cofactor (pyridinium 3-thioamide-5-thiocarboxylic acid mononucleotide) of lactate racemase. Coenzyme A (CoA) stabilizes LarE and forms a CoA-Cys176 mixed disulfide with the protein. This study presents the crystal structure of the LarE/CoA complex, revealing protein interactions with CoA that mimic those for binding ATP. CoA weakly inhibits LarE activity, and the persulfide of CoA is capable of partially regenerating functional LarE from the Dha176 form of the protein. The physiological relevance of this cycling reaction is unclear. A new form of LarE was discovered, an NPN-LarE covalent adduct, explaining prior results in which activation of the lactate racemase apoprotein required only the isolated LarE. The crystal structure of the inactive C176A variant revealed a fold essentially identical to that of wild-type LarE. Additional active site variants of LarE were created and their activities characterized, with all LarE variants analyzed in terms of the structure. Finally, the L. plantarum LarE structure was compared to a homology model of Thermoanaerobacterium thermosaccharolyticum LarE, predicted to contain three cysteine residues at the active site, and to other proteins with a similar fold and multiple active site cysteine residues. These findings suggest that some LarE orthologs may not be sacrificial but instead may catalyze sulfur transfer by using a persulfide mechanism or from a labile site on a [4Fe-4S] cluster at this position.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Lactobacillus plantarum/enzimologia , Racemases e Epimerases/química , Racemases e Epimerases/metabolismo , Enxofre/metabolismo , Proteínas de Bactérias/genética , Catálise , Domínio Catalítico , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Racemases e Epimerases/genéticaRESUMO
Lactate racemase (LarA) of Lactobacillus plantarum contains a novel organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide, but its function in the enzyme mechanism has not been elucidated. This study presents direct evidence that the nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization. No signal was detected by electron paramagnetic resonance spectroscopy for LarA in the absence or presence of substrate, consistent with a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Pyruvate, the predicted intermediate for a PCHT mechanism, was observed in quenched solutions of LarA. A normal substrate kinetic isotope effect ( kH/ kD of 3.11 ± 0.17) was established using 2-α-2H-lactate, further supporting a PCHT mechanism. UV-visible spectroscopy revealed a lactate-induced perturbation of the cofactor spectrum, notably increasing the absorbance at 340 nm, and demonstrated an interaction of the cofactor with the inhibitor sulfite. A crystal structure of LarA provided greater resolution (2.4 Å) than previously reported and revealed sulfite binding to the pyridinium C4 atom of the reduced pincer cofactor, mimicking hydride reduction during a PCHT catalytic cycle. Finally, computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand. In aggregate, these studies provide compelling evidence that the nickel-pincer cofactor acts by a PCHT mechanism.
Assuntos
Proteínas de Bactérias/química , Coenzimas/química , Lactobacillus plantarum/enzimologia , Níquel/química , Compostos Organometálicos/química , Prótons , Racemases e Epimerases/química , Proteínas de Bactérias/genética , Coenzimas/genética , Coenzimas/metabolismo , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Lactobacillus plantarum/genética , Níquel/metabolismo , Compostos Organometálicos/metabolismo , Domínios Proteicos , Racemases e Epimerases/genética , Espectrofotometria UltravioletaRESUMO
Cytochromes c require covalent attachment of heme via two thioether bonds at conserved CXXCH motifs, a process accomplished in prokaryotes by eight integral membrane proteins (CcmABCDEFGH), termed System I. Heme is trafficked from inside the cell to outside (via CcmABCD) and chaperoned (holoCcmE) to the cytochrome c synthetase (CcmF/H). Purification of key System I pathway intermediates allowed the determination of heme redox potentials. The data support a model whereby heme is oxidized to form holoCcmE and subsequently reduced by CcmF/H for thioether formation, with Fe(2+) being required for attachment to CXXCH. Results provide insight into mechanisms for the oxidation and reduction of heme in vivo.
Assuntos
Citocromos c/metabolismo , Heme/química , Heme/metabolismo , Liases/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Escherichia coli/metabolismo , Immunoblotting , Oxirredução , Transporte ProteicoRESUMO
A novel organometallic cofactor, nickel pyridinium-3,5-dithiocarboxylic acid mononucleotide, was recently discovered in lactate racemase (LarA) of Lactobacillus plantarum. This review summarizes the substantial progress made in uncovering the function of this cofactor as a transient hydride acceptor in the LarA mechanism. The latest developments related to cofactor biosynthesis reveal insights into a pathway in which LarB serves as a nicotinic acid adenine dinucleotide hydrolase/carboxylase, LarE acts as a sacrificial sulfur transferase, and LarC functions as a nickel insertase, forming the nickel-pincer nucleotide cofactor that becomes covalently tethered to LarA in some bacteria. Bioinformatic studies reveal a widespread occurrence of larA, larB, larC, and larE orthologs in microorganisms, and additional roles for the cofactor are considered.