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Abstract The antibiotic moenomycin A is a phosphoglycerate derivative with a C 25 -moenocinyl chain and a branched oligosaccharide. Formation of the C 25 -chain is catalyzed by the enzyme MoeN5 with geranyl pyrophosphate (GPP) and the sugar-linked 2- Z,E -farnesyl-3-phosphoglycerate (FPG) as its substrates. Previous complex crystal structures with GPP and long-chain alkyl glycosides suggested that GPP binds to the S1 site in a similar way as in most other α-helical prenyltransferases (PTs), and FPG is likely to assume a bent conformation in the S2 site. However, two FPG derivatives synthesized in the current study were found in the S1 site rather than S2 in their complex crystal structures with MoeN5. Apparently S1 is the preferred site for prenyl-containing ligand, and S2 binding may proceed only after S1 is occupied. Thus, like most trans -type PTs, MoeN5 may employ a sequential ionization-condensation-elimination mechanism that involves a carbocation intermediate. Highlights • The complex crystal structures of MoeN5/FQ1 and MoeN5/FQ2 were determined. • FQ1 and FQ2 were found in the other binding site for geranyl pyrophosphate (GPP). • The ordered binding suggests a sequential mechanism starting with GPP ionization. • Docking of the Sso7d tag on MoeN5 helped with crystallization. [ABSTRACT FROM AUTHOR]

Pyridoxine-5′-phosphate oxidase catalyzes the oxidation of either the C4′ alcohol group or amino group of the two substrates pyridoxine 5′-phosphate and pyridoxamine 5′-phosphate to an aldehyde, forming pyridoxal 5′-phosphate. A hydrogen atom is removed from C4′ during the oxidation and a pair of electrons is transferred to tightly bound FMN. A new crystal form of the enzyme in complex with pyridoxal 5′-phosphate shows that the N-terminal segment of the protein folds over the active site to sequester the ligand from solvent during the catalytic cycle. Using (4′R)-[3H]PMP as substrate, nearly 100 % of the radiolabel appears in water after oxidation to pyridoxal 5′-phosphate. Thus, the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4′ carbon atom of pyridoxamine 5′-phosphate. Site mutants were made of all residues at the active site that interact with the oxygen atom or amine group on C4′ of the substrates. Other residues that make interactions with the phosphate moiety of the substrate were mutated. The mutants showed a decrease in affinity, but exhibited considerable catalytic activity, showing that these residues are important for binding, but play a lesser role in catalysis. The exception is Arg197, which is important for both binding and catalysis. The R197 M mutant enzyme catalyzed removal of the proS hydrogen atom from (4′R)-[3H]PMP, showing that the guanidinium side-chain plays an important role in determining stereospecificity. The crystal structure and the stereospecificity studies suggests that the pair of electrons on C4′ of the substrate are transferred to FMN as a hydride ion. [ABSTRACT FROM AUTHOR]

Bacterial wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising the transmembrane and ATPase subunits TagG and TagH. Here the dimeric structure of the N-terminal domain of TagH (TagH-N) was solved by single-wavelength anomalous diffraction using a selenomethionine-containing crystal, which shows an ATP-binding cassette (ABC) architecture with RecA-like and helical subdomains. Besides significant structural differences from other ABC transporters, a prominent patch of positively charged surface is seen in the center of the TagH-N dimer, suggesting a potential binding site for the glycerol phosphate chain of WTA. The ATPase activity of TagH-N was inhibited by clodronate, a bisphosphonate, in a non-competitive manner, consistent with the proposed WTA-binding site for drug targeting. • The dimeric structure of the N-terminal RecA-like domain of TagH was solved. • Positively charged surface in the center suggests a potential binding site for WTA. • The ATPase activity was inhibited by clodronate in a non-competitive manner. [ABSTRACT FROM AUTHOR]