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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

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]

Examines the pyruvate site of pyruvate phosphate dikinase for crystal structure of the enzyme-phosphonopyruvate complex and mutant analysis. Association of magnesium-bound phosphonopyruvate with alpha and beta-barrels; Resemblance of C-termini in beta-strands; Formation of remote substrate binding sites.

Examines the serine-beta-lactamases hydrolyze beta-lactam antibiotics in a reaction via an acylenyme intermediate. Measurement of the firmness of the crystal structure of beta-lactamase; Location of the thiadolizine ring of the cleaved penicillin; Trends of the three modes of binding.

Studies the elimination of the hydrolytic water molecule in a class A beta lactamase. Redefinition of the crystal structures of mutant enzymes; Displacement of water molecules in the side chain of Gln 170; Rate of acylation of the native enzyme.