Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions ...by sialic acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid (Pse)). The fourth enzymatic step in the pseudaminic acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-L-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 Å resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His¹⁷-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His¹⁷ functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C-O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.
Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. The heptasaccharide produced by its pgl system is attached to protein Asn through its terminal ...2,4-diacetamido-2,4,6-trideoxy-d-Glc (QuiNAc4NAc or N,N‘-diacetylbacillosamine) moiety. The crucial, last part of this sugar's synthesis is the acetylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-d-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms, refined to 1.8 and 1.75 Å resolution, respectively. PglD is a trimer of subunits each comprised of two domains, an N-terminal α/β-domain and a C-terminal left-handed β-helix. Few structural differences accompany CoA binding, except in the C-terminal region following the β-helix (residues 189−195), which adopts an extended structure in the unbound form and folds to extend the β-helix upon binding CoA. Computational molecular docking suggests a different mode of nucleotide−sugar binding with respect to the acetyl-CoA donor, with the molecules arranged in an “L-shape”, compared with the “in-line” orientation in related enzymes. Modeling indicates that the oxyanion intermediate would be stabilized by the NH group of Gly143‘, with His125‘ the most likely residue to function as a general base, removing H+ from the amino group prior to nucleophilic attack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed the importance of His125‘, Glu124‘, and Asn118. We conclude that Asn118 exerts its function by stabilizing the intricate hydrogen bonding network within the active site and that Glu124‘ may function to increase the pK a of the putative general base, His125‘.
The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and ...sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters.
Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. The heptasaccharide produced by its pgl system is attached to protein Asn through its terminal ...2,4-diacetamido-2,4,6- trideoxy-D-Glc (QuiNAc4NAc or N,N'-diacetylbacillosamine) moiety. The crucial, last part of this sugar's synthesis is the acetylation of UDP-2- acetamido-4-amino-2,4,6-trideoxy-D-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms, refined to 1.8 and 1.75 Aa resolution, respectively. PglD is a trimer of subunits each comprised of two domains, an N-terminal alpha/beta2-domain and a C-terminal left-handed beta2-helix. Few structural differences accompany CoA binding, except in the C-terminal region following the beta2-helix (residues 189-195), which adopts an extended structure in the unbound form and folds to extend the beta2-helix upon binding CoA. Computational molecular docking suggests a different mode of nucleotide-sugar binding with respect to the acetyl-CoA donor, with the molecules arranged in an "L-shape", compared with the "in-line" orientation in related enzymes. Modeling indicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' the most likely residue to function as a general base, removing H super(+) from the amino group prior to nucleophilic attack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed the importance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing the intricate hydrogen bonding network within the active site and that Glu124' may function to increase the pK sub(a) of the putative general base, His125'.
The human protein p54
nrb and its mouse homolog NonO have been implicated in a variety of nuclear processes including transcription, pre-mRNA processing, nuclear retention of edited RNA and DNA ...relaxation. We have identified p54
nrb as an antigen of the phosphodependent monoclonal antibodies CC-3 and MPM-2 and shown that this protein is phosphorylated on multiple sites during mitosis. The use of the cyclin-dependent protein kinase inhibitor roscovitine and immunodepletion studies with an anti-cyclin B1 antibody established that Cdk1 was responsible for the phosphorylation of the carboxy-terminal extremity of p54
nrb whereas a different kinase appeared to be involved in the generation of CC-3 epitope(s) in the amino-terminal moiety of the protein. Like many CC-3 and MPM-2 antigens, we show that p54
nrb is a target of the peptidylprolyl isomerase Pin1, suggesting that it may be regulated by phosphorylation-dependent conformational changes as many other nuclear proteins upon entry into mitosis. In addition, site-directed mutagenesis indicated that the interaction of Pin1 with p54
nrb was mediated by three threonine residues located in the proline-rich carboxy-terminal extremity of the protein. Our results also showed that Pin1 binding was favored when at least two of the three threonine residues were phosphorylated, suggesting a regulation mechanism based on multisite phosphorylation.
The human protein p54nrb and its mouse homolog NonO have been implicated in a variety of nuclear processes including transcription, pre-mRNA processing, nuclear retention of edited RNA and DNA ...relaxation. We have identified p54nrb as an antigen of the phosphodependent monoclonal antibodies CC-3 and MPM-2 and shown that this protein is phosphorylated on multiple sites during mitosis. The use of the cyclin-dependent protein kinase inhibitor roscovitine and immunodepletion studies with an anti-cyclin B1 antibody established that Cdk1 was responsible for the phosphorylation of the carboxy-terminal extremity of p54nrb whereas a different kinase appeared to be involved in the generation of CC-3 epitope(s) in the amino-terminal moiety of the protein. Like many CC-3 and MPM-2 antigens, we show that p54nrb is a target of the peptidylprolyl isomerase Pin1, suggesting that it may be regulated by phosphorylation-dependent conformational changes as many other nuclear proteins upon entry into mitosis. In addition, site-directed mutagenesis indicated that the interaction of Pin1 with p54nrb was mediated by three threonine residues located in the proline-rich carboxy-terminal extremity of the protein. Our results also showed that Pin1 binding was favored when at least two of the three threonine residues were phosphorylated, suggesting a regulation mechanism based on multisite phosphorylation.
There is a considerable interest in the modification of existing antibiotics to generate new antimicrobials. Glycopeptide antibiotics (GPAs) are effective against serious Gram-positive bacterial ...pathogens including methicillin-resistant
Staphylococcus aureus. However, resistance to these antibiotics is becoming a serious problem requiring new strategies. We show that the
Amycolatopsis orientalis (
S)-adenosyl-
L-methionine-dependent methyltransferase MtfA, from the vancomycin-class GPA chloroeremomycin biosynthetic pathway, catalyzes in vivo and in vitro methyl transfer to generate methylated GPA derivatives of the teicoplanin class. The crystal structure of MtfA complexed with (
S)-adenosyl-
L-methionine, (
S)-adenosylhomocysteine, or sinefungin inhibitor, coupled with mutagenesis, identified His228 as a likely general base required for methyl transfer to the N terminus of the glycopeptide. Computational docking and molecular dynamics simulations were used to model binding of demethyl-vancomycin aglycone to MtfA. These results demonstrate its utility as a tool for engineering methylated analogs of GPAs.
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