Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate

In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes

Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism

Kinases are ubiquitous enzymes that are pivotal to many biochemical processes. There are contrasting views on the phosphoryl-transfer mechanism in propionate kinase, an enzyme that reversibly transfers a phosphoryl group from propionyl phosphate to ADP in the final step of non-oxidative catabolism of l-threonine to propionate. Here, X-ray crystal structures of propionate- and nucleotide-bound Salmonella typhimurium propionate kinase are reported at 1.8-2.0 Å resolution. Although the mode of nucleotide binding is comparable to those of other members of the ASKHA superfamily, propionate is bound at a distinct site deeper in the hydrophobic pocket defining the active site. The propionate carboxyl is at a distance of ∼5 Å from the γ-phosphate of the nucleotide, supporting a direct in-line transfer mechanism. The phosphoryl-transfer reaction is likely to occur via an associative SN2-like transition state that involves a pentagonal bipyramidal structure with the axial positions occupied by the nucleophile of the substrate and the O atom between the β- and the γ-phosphates, respectively. The proximity of the strictly conserved His175 and Arg236 to the carboxyl group of the propionate and the γ-phosphate of ATP suggests their involvement in catalysis. Moreover, ligand binding does not induce global domain movement as reported in some other members of the ASKHA superfamily. Instead, residues Arg86, Asp143 and Pro116-Leu117-His118 that define the active-site pocket move towards the substrate and expel water molecules from the active site. The role of Ala88, previously proposed to be the residue determining substrate specificity, was examined by determining the crystal structures of the propionate-bound Ala88 mutants A88V and A88G. Kinetic analysis and structural data are consistent with a significant role of Ala88 in substrate-specificity determination. The active-site pocket-defining residues Arg86, Asp143 and the Pro116-Leu117-His118 segment are also likely to contribute to substrate specificity

A Tail-Based Mechanism Drives Nucleosome Demethylation by the LSD2/NPAC Multimeric Complex

Summary: LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes. : Through biophysical, biochemical, and structural studies, including cryo-EM, Marabelli et al. describe how NPAC promotes LSD2 productive interaction with the nucleosome in a rapid and flexible manner. Their findings provide a molecular mechanism for LSD2 activity in the context of H3K4me2 demethylation during Pol II transcriptional elongation. Keywords: histone demethylation, cryoelectron microscopy, chromatin reader, flavoenzyme, epigenetics, evolution of protein function, molecular recognitio

High Potency of a Bivalent Human VH Domain in SARS-CoV-2 Animal Models

Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VHFc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic

Preliminary X-ray crystallographic studies on acetate kinase (AckA) from Salmonella typhimurium in two crystal forms

Acetate kinase (AckA) catalyzes the reversible transfer of a phosphate group from acetyl phosphate to ADP, generating acetate and ATP, and plays a central role in carbon metabolism. In the present work, the gene corresponding to AckA from Salmonella typhimurium (StAckA) was cloned in the IPTG-inducible pRSET C vector, resulting in the attachment of a hexahistidine tag to the N-terminus of the expressed enzyme. The recombinant protein was overexpressed, purified and crystallized in two different crystal forms using the microbatch-under-oil method. Form I crystals diffracted to 2.70 angstrom resolution when examined using X-rays from a rotating-anode X-ray generator and belonged to the monoclinic space group C2, with unit-cell parameters a = 283.16, b = 62.17, c = 91.69 angstrom, beta = 93.57 degrees. Form II crystals, which diffracted to a higher resolution of 2.35 angstrom on the rotating-anode X-ray generator and to 1.90 angstrom on beamline BM14 of the ESRF, Grenoble, also belonged to space group C2 but with smaller unit-cell parameters (a = 151.01, b = 78.50, c = 97.48 angstrom, beta = 116.37 degrees). Calculation of Matthews coefficients for the two crystal forms suggested the presence of four and two protomers of StAckA in the asymmetric units of forms I and II, respectively. Initial phases for the form I diffraction data were obtained by molecular replacement using the coordinates of Thermotoga maritima AckA (TmAckA) as the search model. The form II structure was phased using a monomer of form I as the phasing model. Inspection of the initial electron-density maps suggests dramatic conformational differences between residues 230 and 300 of the two crystal forms and warrants further investigation

Tyrosine 66 of Pepper vein banding virus genome-linked protein is uridylylated by RNA-dependent RNA polymerase

Pepper vein banding virus (PVBV), a member of the genus potyvirus, is a single-stranded positive-sense RNA virus and it primarily infects plants of the family Solanaceae. Genome organization and gene expression strategy of the polyviruses are similar to the picomaviruses, although they infect widely different hosts and have distinctly different morphologies. The genomic RNA of PVBV has a viralgenome-linked protein (VPg) at the 5'-terminus and a poly(A) tail atthe 3'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV NIb and VPg were over-expressed in Escherichia coli and purified under non-denaturing conditions. PVBV NIb was found to be active as polymerase and it could uridylylate the VPg in a template independent manner. N- and C-terminal deletion analysis of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation. The amino acid residue uridylylated by PVBVNIb was identified to be Tyr 66 by site-directed mutagenesis. It is possible that in potyviruses, replication begins with uridylylation of VPg which acts as primer for progeny RNA synthesis

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4 A resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coil CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes. (C) 2010 Elsevier Inc. All rights reserved

Tyrosine 66 of Pepper vein banding virus genome-linked protein is uridylylated by RNA-dependent RNA polymerase

Pepper vein banding virus (PVBV), a member of the genus potyvirus, is a single-stranded positive-sense RNA virus and it primarily infects plants of the family Solanaceae. Genome organization and gene expression strategy of the polyviruses are similar to the picomaviruses, although they infect widely different hosts and have distinctly different morphologies. The genomic RNA of PVBV has a viralgenome-linked protein (VPg) at the 5'-terminus and a poly(A) tail atthe 3'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV NIb and VPg were over-expressed in Escherichia coli and purified under non-denaturing conditions. PVBV NIb was found to be active as polymerase and it could uridylylate the VPg in a template independent manner. N- and C-terminal deletion analysis of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation. The amino acid residue uridylylated by PVBVNIb was identified to be Tyr 66 by site-directed mutagenesis. It is possible that in potyviruses, replication begins with uridylylation of VPg which acts as primer for progeny RNA synthesis

Structural and mechanistic investigations on <it>Salmonella typhimurium</it> acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface

<p>Abstract</p> <p>Background</p> <p>Bacteria such as <it>Escherichia coli</it> and <it>Salmonella typhimurium</it> can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism.</p> <p>Results</p> <p>Here we report kinetic characterization of <it>S. typhimurium</it> AckA (<it>St</it>AckA) and structures of its unliganded (Form-I, 2.70 Å resolution) and citrate-bound (Form-II, 1.90 Å resolution) forms. The enzyme showed broad substrate specificity with <it>k</it>_<it>cat</it>/<it>K</it>_<it>m</it> in the order of acetate > propionate > formate. Further, the <it>K</it>_<it>m</it> for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (<it>i.e.</it> ATP synthesis) more efficiently. ATP and Mg²⁺ could be substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP) and divalent cations (Mn²⁺ and Co²⁺), respectively. Form-I <it>St</it>AckA represents the first structural report of an unliganded AckA. <it>St</it>AckA protomer consists of two domains with characteristic βββαβαβα topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound <it>Methanosarcina thermophila</it> AckA (<it>Mt</it>AckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II <it>St</it>AckA structure showed a drastic change in the conformation of residues 230–300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II <it>St</it>AckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I <it>St</it>AckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes<it>.</it></p> <p>Conclusions</p> <p>The biochemical and structural characterization of <it>St</it>AckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of <it>St</it>AckA led to identification of a putative ligand-binding pocket at the dimeric interface of <it>St</it>AckA with implications for enzymatic function.</p

Tyrosine 66 of Pepper vein banding virus genome-linked protein is uridylylated by RNA-dependent RNA polymerase

Pepper vein banding virus (PVBV), a member of the genus potyvirus, is a single-stranded positive-sense RNA virus and it primarily infects plants of the family Solanaceae. Genome organization and gene expression strategy of the potyviruses are similar to the picornaviruses, although they infect widely different hosts and have distinctly different morphologies. The genomic RNA of PVBV has a viral genome-linked protein (VPg) at the 5'-terminus and a poly(A) tail at the 3'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV NIb and VPg were over-expressed in Escherichia coli and purified under non-denaturing conditions. PVBV NIb was found to be active as polymerase and it could uridylylate the VPg in a template independent manner. N- and C-terminal deletion analysis of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation. The amino acid residue uridylylated by PVBV NIb was identified to be Tyr 66 by site-directed mutagenesis. It is possible that in potyviruses, replication begins with uridylylation of VPg which acts as primer for progeny RNA synthesis