Sphingomyelinases C are enzymes that catalyze the hydrolysis of sphingomyelin in biological membranes to ceramide and phosphorylcholine. Various pathogenic bacteria produce secreted neutral sphingomyelinases C that act as membrane-damaging virulence factors. Mammalian neutral sphingomyelinases C, which display sequence homology to the bacterial enzymes, are involved in sphingolipid metabolism and signaling. This article describes the first structure to be determined for a member of the neutral sphingomyelinase C family, SmcL, from the intracellular bacterial pathogen Listeria ivanovii. The structure has been refined to 1.9-Å resolution with phases derived by single isomorphous replacement with anomalous scattering techniques from a single iridium derivative. SmcL adopts a DNase I-like fold, and is the first member of this protein superfamily to have its structure determined that acts as a phospholipase. The structure reveals several unique features that adapt the protein to its phospholipid substrate. These include large hydrophobic -hairpin and hydrophobic loops surrounding the active site that may bind and penetrate the lipid bilayer to position sphingomyelin in a catalytically competent position. The structure also provides insight into the proposed general base/acid catalytic mechanism, in which His-325 and His-185 play key roles. Sphingomyelinases C (SMases C)4 (EC 3.1.4.12) are phosphodiesterases that catalyze the hydrolysis of the membrane phospholipid sphingomyelin (SM) at the aqueous:lipid interface, generating ceramide and phosphorylcholine. Several types of enzymes with SMase C activity have been identified in eukaryotes and prokaryotes. Eukaryotic SMases C have been classified according to their pH optima and are known as acid SMase (1), alkaline SMase (2), and neutral SMase (nSMase) (3, 4). In prokaryotes, some broad specificity phosphatidylcholine phospholipases C display SM hydrolyzing activity (5, 6) but a number of pathogenic bacteria, such as Staphylococcus aureus (-toxin (7)), Bacillus cereus (8), Leptospira interrogans (9), and Listeria ivanovii (10), produce SM-specific phospholipases. These bacterial SMases C share sequence homology with the eukaryotic nSMases, and all currently available data suggests that eukaryotic nSMases and bacterial SMases C (henceforth bacterial nSMases) share a similar catalytic mechanism and overall structure (11,12). A sequence alignment of selected members of the nSMase family is presented in Fig. 1. In contrast to the eukaryotic and bacterial nSMases there is no identifiable sequence conservation within the other types of SMase C. Additionally, these enzymes utilize different catalytic mechanisms, and are predicted to be structurally unrelated to nSMases.Mammalian nSMases are thought to play a key role in sphingolipid metabolism and there is increasing evidence implicating SM metabolites in cell signaling, cell proliferation, and apoptosis (13-16). Two human nSMases have been cloned, nSMase1 (3) and nSMase2 (4). Sequence analysis of these proteins and other e...
Both monomeric and dimeric NADP-dependent isocitrate dehydrogenase (IDH) catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxaloglutarate. Monomeric NADP-specific IDHs have been identified from about 50 different bacteria, whereas, dimeric NADP-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed the phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP-IDHs. This is done to get an idea of evolutionary relationship. It is important to solve the structures of IDH from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate-bound (NADP or isocitrate) NADP-dependent monomeric IDH from Azotobacter Vinelandii (AvIDH) have been solved. Here, we are reporting for the first time the substrate free structure of monomeric IDH from Corynebacterium glutamicum (CgIDH) White et al. [6]. A structural change was postulated because SCOT is more readily inactivated by DTNB binding when the enzyme is bound to CoA. The specific cysteine being labeled was identified by Rochet [7] to be Cys28. We have studied the importance of this residue using site directed mutagenesis, kinetics as well as X-ray crystallography. The mutants constructed are C28S, C28A and C28W. C28A and C28S have be crystallized in P21 with dimensions a=63 Å, b=263 Å, c=59 Å, =110º and both have diffracted to better then 2.3 Å.[1] Stern et al., J. Biol. Chem., 1956, 221 The 2.2 Å resolution crystal structure of the enzyme phosphoenolpyruvate carboxykinase (PCK) from the bacterium Anaerobiospirillum succiniciproducens complexed with ATP, Mg 2+ , Mn 2+ and the transition state analogue oxalate has been solved. The 2.4 Å resolution native structure of A. succiniciproducens PCK has also been determined. It has been found that upon binding of substrate, PCK undergoes a conformational change. Two domains of the molecule fold towards each other, with the substrates and metal ions held in a cleft formed between the two domains. This domain movement is believed to accelerate the reaction PCK catalyzes by forcing bulk solvent molecules out of the active site. Although the crystal structure of A. succiniciproducens PCK with bound substrate and metal ions is related to the structures of PCK from Escherichia coli and Trypanosoma cruzi, it is the first crystal structure from this class of enzymes that clearly shows an important surface loop (residues 383 to 397) from the C-terminal domain, hydrogen bonding with the peptide backbone of the active site residue Arg60. The interaction between the surface loop and the active site backbone, which is a parallel -sheet, seems to be a feature unique of A. succiniciproducens PCK. The association between the loop and the active site is the third type of interaction found in PCK that is thought to play a part in the domain closure. This loop also appears to help accelerate catalysis by functioning as a 'lid' that shields water molecules from the active site. Bristol, Langford, Bristol, ...
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