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The crystal structure studies of a second conformational state of Clostridium symbiosum pyruvate phosphate dikinase (PPDK) are reported. The studies confirm the swivel motion of the His domain adjacent to the phosphoenolpyruvate (PEP)-binding domain.

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP): sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the [Mg.sup.2+]-dependent phosphorylation of enzyme I (El) by PEP. Crystals of Escherichia coli El were obtained by mixing the protein with [Mg.sup.2+] and PEP, followed by oxalate, an El inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by [Mg.sup.2+] and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189~P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an [alpha]-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an El fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189~P and His-15 are appropriately stationed for an in-line phosphotransfer reaction. sugar transport | phosphorylation | x-ray crystallography

Structural analysis of the enzyme-substrate complex reveals that phosphonopyruvate binds in the alpha/beta-barrel's central channel of the Beta-strands C-termini. The structure resembles that of pyruvate kinase but differes in the acid/base catalysis function.

Research on a mutant form of beta lactamase from Staphylococcus aureus shows that substrate specificity is not determined at the acyl-enzyme state. This was the conclusion of researchers who studied the binding modes of benzylpenicillin and cephaloridine.

Two site-directed mutant enzymes of the class A beta-lactamase from Staphylococcus aureus PC1 were produced to block the site occupied by a hydrolytic water molecule in the native enzyme. The crystal structures of these two mutant enzymes, N170Q and N170M, were determined. Results indicated that the water site is preserved and that Met170 occupies part of the space that is needed for substrate binding. The short contacts between the substrate and the enzyme may cause structure perturbation and inactivation.

The crystal structure of pyruvate phosphate dikinase, a histidyl multiphosphotransfer enzyme that synthesizes adenosine triphosphate, reveals a three-domain molecule in which the phosphohistidine domain is flanked by the nucleotide and the phosphoenolpyruvate/pyruvate domains, with the two substrate binding sites [approximately equal to]45 [Angstrom] apart. The modes of substrate binding have been deduced by analogy to D-Ala-D-Ala ligase and to pyruvate kinase. Coupling between the two remote active sites is facilitated by two conformational states of the phosphohistidine domain. While the crystal structure represents the state of interaction with the nucleotide, the second state is achieved by swiveling around two flexible peptide linkers. This dramatic conformational transition brings the phosphocarrier residue in close proximity to phosphoenolpyruvate/pyruvate. The swiveling-domain paradigm provides an effective mechanism for communication in complex multidomain/multiactive site proteins.

Phosphonates allow certain organisms to thrive in otherwise hostile environments, and 2-aminoethylphosphonate (AEP) is a precursor of many cellular phosphonates. AEP transaminase (AEPT) is an enzyme essential to phosphonate synthesis and degradation pathways. The crystal structure of AEP transaminase was determined by multiwavelength anomalous diffraction of 66 selenium atoms. The refined structure at 2.2 Å resolution revealed an overall fold and active site location similar to those of the dimeric, two-domain structure of type I aminotransferases. The active site contains a cofactor, pyridoxal 5'-phosphate (PLP), and the product phosphonoacetaldehyde. Comparison with other type I aminotransferase structures shows that the PLP-protein interactions are conserved. Modeling of bound substrates and products reveals the structural basis for AEP recognition and the stereospecificity of proton elimination at the α-carbon and indicates conformational changes along the reaction pathway. Phosphonates allow certain organisms to thrive in otherwise hostile environments, and 2-aminoethylphosphonate (AEP) is a precursor of many cellular phosphonates. AEP transaminase (AEPT) is an enzyme essential to phosphonate synthesis and degradation pathways. The crystal structure of AEP transaminase was determined by multiwavelength anomalous diffraction of 66 selenium atoms. The refined structure at 2.2 Å resolution revealed an overall fold and active site location similar to those of the dimeric, two-domain structure of type I aminotransferases. The active site contains a cofactor, pyridoxal 5'-phosphate (PLP), and the product phosphonoacetaldehyde. Comparison with other type I aminotransferase structures shows that the PLP-protein interactions are conserved. Modeling of bound substrates and products reveals the structural basis for AEP recognition and the stereospecificity of proton elimination at the α-carbon and indicates conformational changes along the... [ABSTRACT FROM AUTHOR]