scholarly journals Crystal Structure of the PdxY Protein from Escherichia coli

2004 ◽  
Vol 186 (23) ◽  
pp. 8074-8082 ◽  
Author(s):  
Martin K. Safo ◽  
Faik N. Musayev ◽  
Sharyn Hunt ◽  
Martino L. di Salvo ◽  
Neel Scarsdale ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Binding Site ◽  
Sequence Homology ◽  
Active Site ◽  
Pyridoxal Phosphate ◽  
Protein A ◽  
Detailed Comparison ◽  
Protein Product ◽  
Atp Binding Site

ABSTRACT The crystal structure of Escherichia coli PdxY, the protein product of the pdxY gene, has been determined to a 2.2-Å resolution. PdxY is a member of the ribokinase superfamily of enzymes and has sequence homology with pyridoxal kinases that phosphorylate pyridoxal at the C-5′ hydroxyl. The protein is a homodimer with an active site on each monomer composed of residues that come exclusively from each respective subunit. The active site is filled with a density that fits that of pyridoxal. In monomer A, the ligand appears to be covalently attached to Cys122 as a thiohemiacetal, while in monomer B it is not covalently attached but appears to be partially present as pyridoxal 5′-phosphate. The presence of pyridoxal phosphate and pyridoxal as ligands was confirmed by the activation of aposerine hydroxymethyltransferase after release of the ligand by the denaturation of PdxY. The ligand, which appears to be covalently attached to Cys122, does not dissociate after denaturation of the protein. A detailed comparison (of functional properties, sequence homology, active site and ATP-binding-site residues, and active site flap types) of PdxY with other pyridoxal kinases as well as the ribokinase superfamily in general suggested that PdxY is a member of a new subclass of the ribokinase superfamily. The structure of PdxY also permitted an interpretation of work that was previously published about this enzyme.

scholarly journals Crystal Structure of Pyridoxal Kinase from the Escherichia coli pdxK Gene: Implications for the Classification of Pyridoxal Kinases

2006 ◽  
Vol 188 (12) ◽  
pp. 4542-4552 ◽  
Author(s):  
Martin K. Safo ◽  
Faik N. Musayev ◽  
Martino L. di Salvo ◽  
Sharyn Hunt ◽  
Jean-Baptiste Claude ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Active Site ◽  
Pyridoxal Phosphate ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
E Coli ◽  
Binary Complexes ◽  
Significant Conformational Change

ABSTRACT The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-Å resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4′ substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4′ of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.

scholarly journals Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii

2017 ◽  
Vol 73 (12) ◽  
pp. 651-656 ◽  
Author(s):  
Taichi Mizobuchi ◽  
Risako Nonaka ◽  
Motoki Yoshimura ◽  
Katsumasa Abe ◽  
Shouji Takahashi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Active Site ◽  
Metal Binding ◽  
Bivalve Mollusc ◽  
Active Site Residues ◽  
X Ray ◽  
Aspartate Racemase ◽  
Scapharca Broughtonii

Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

scholarly journals Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli

FEBS Letters ◽  
1996 ◽  
Vol 398 (2-3) ◽  
pp. 151-154 ◽  
Author(s):  
Dong Hun Shin ◽  
Soon Ji Yoo ◽  
Yoon Kyung Shim ◽  
Jae Hong Seol ◽  
Man-Sik Kang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Mutational Analysis ◽  
Atp Binding ◽  
Atp Binding Site

scholarly journals The crystal structure of the Rv0301-Rv0300 VapBC-3 toxin-antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA-binding site

2012 ◽  
Vol 21 (11) ◽  
pp. 1754-1767 ◽  
Author(s):  
Andrew B. Min ◽  
Linda Miallau ◽  
Michael R. Sawaya ◽  
Jeff Habel ◽  
Duilio Cascio ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Active Site ◽  
Rna Binding

Selectivity of the surface binding site (SBS) on barley starch synthase I

Biologia ◽  
2014 ◽  
Vol 69 (9) ◽  
Author(s):  
Casper Wilkens ◽  
Jose Cuesta-Seijo ◽  
Monica Palcic ◽  
Birte Svensson
Keyword(s):  
Crystal Structure ◽  
Surface Plasmon Resonance ◽  
Binding Site ◽  
Active Site ◽  
Mutational Analysis ◽  
Starch Synthase ◽  
Surface Binding ◽  
Binding Studies ◽  
A Chain ◽  
Surface Binding Site

AbstractStarch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was seen, which was found by mutational analysis to be essential for the activity of HvSSI on glycogen. We now show in binding studies using surface plasmon resonance that HvSSI has no detectable affinity for malto-triose and -tetraose, but clearly binds maltopentaose, -hexaose, -heptaose (M7) and β-cyclodextrin (β-CD) albeit with a measurable K D for only β-CD and M7. Moreover, an HvSSI SBS mutant F538A lost the ability to bind β-CD and maltooligosaccharides. This behaviour suggests that a chain in the α-glucan molecule (amylopectin) that is undergoing extension attaches itself at the SBS and that the active site itself, likely working on a different end chain, has low affinity for both substrate and product.

scholarly journals Crystal structures of two monomeric triosephosphate isomerase variants identifiedviaa directed-evolution protocol selecting forL-arabinose isomerase activity

2016 ◽  
Vol 72 (6) ◽  
pp. 490-499
Author(s):  
Mirja Krause ◽  
Tiila-Riikka Kiema ◽  
Peter Neubauer ◽  
Rik K. Wierenga
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Directed Evolution ◽  
Crystal Structures ◽  
Active Site ◽  
Triosephosphate Isomerase ◽  
Point Mutations ◽  
Van Der Waals Interactions ◽  
Side Chain ◽  
Arabinose Isomerase

The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using anEscherichia coliL-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of theEscherichia coliselection strain in medium supplemented with 40 mML-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.

scholarly journals The nucleotide-binding domain of the Zn2+-transporting P-type ATPase from Escherichia coli carries a glycine motif that may be involved in binding of ATP

2004 ◽  
Vol 377 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Juha OKKERI ◽  
Liisa LAAKKONEN ◽  
Tuomas HALTIA
Keyword(s):  
Escherichia Coli ◽  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Molecular Model ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Disease Mutations ◽  
Disease Protein ◽  
P Type

In P-type ATPases, the nucleotide-binding (N) domain is located in the middle of the sequence which folds into the phosphorylation (P) domain. The N domain of ZntA, a Zn2+-translocating P-type ATPase from Escherichia coli, is approx. 13% identical with the N domain of sarcoplasmic reticulum Ca2+-ATPase. None of the Ca2+-ATPase residues involved in binding of ATP are found in ZntA. However, the sequence G503SGIEAQV in the N domain of ZntA resembles the motif GxGxxG, which forms part of the ATP-binding site in protein kinases. This motif is also found in Wilson disease protein where several disease mutations cluster in it. In the present work, we have made a set of disease mutation analogues, including the mutants G503S (Gly503→Ser), G505R and A508F of ZntA. At low [ATP], these mutant ATPases are poorly phosphorylated. The phosphorylation defect of the mutants G503S and G505R can, however, be partially (G503S) or fully (G505R) compensated for by using a higher [ATP], suggesting that these mutations lower the affinity for ATP. In all three mutant ATPases, phosphorylation by Pi has become less sensitive to the presence of ATP, also consistent with the proposal that the Gly503 motif plays a role in ATP binding. In order to test this hypothesis, we have modelled the N domain of ZntA using the sarcoplasmic reticulum Ca2+-ATPase structure as a template. In the model, the Gly503 motif, as well as the residues Glu470 and His475, are located in the proximity of the ATP-binding site. In conclusion, the mutagenesis data and the molecular model are consistent with the idea that the two loops carrying the residues Glu470, His475, Gly503 and Gly505 play a role in ATP binding and activation.

scholarly journals Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase

FEBS Letters ◽  
1988 ◽  
Vol 235 (1-2) ◽  
pp. 189-193 ◽  
Author(s):  
Peter M. Jordan ◽  
Martin J. Warren ◽  
Howard J. Williams ◽  
Neal J. Stolowich ◽  
Charles A. Roessner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Active Site ◽  
Cysteine Residue ◽  
Porphobilinogen Deaminase
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