Abstract7-Azaindole is the chromophoric moiety of 7-azatryptophan, which is an alternative to tryptophan as an optical probe of protein structure and dynamics. The great power of the 7-azaindole chromophore is that it is red shifted both in absorption and emission from tryptophan, that its fluorescence decay is single exponential in water under appropriate conditions, and that its emission is sensitive to solvent. In addition, 7-azatryptophan can be incorporated into synthetic peptides and bacterial protein. In this article, the interactions of 7-azaindole with its environment are discussed. Special attention is directed to the difference in its fluorescence properties in water as opposed to nonaqueous solvents. The sensitivity of 7-azaindole to its environment is demonstrated and then exploited by studying it and its analogs in peptides and in complexes with larger proteins containing many tryptophan residues. Departments of Chemistry and Biochemistry and Biophysics Iowa State UniVersity, Ames, Iowa 50011ReceiVed: October 1, 1996; In Final Form: January 7, 1997 X 7-Azaindole is the chromophoric moiety of 7-azatryptophan, which is an alternative to tryptophan as an optical probe of protein structure and dynamics. The great power of the 7-azaindole chromophore is that it is red shifted both in absorption and emission from tryptophan, that its fluorescence decay is single exponential in water under appropriate conditions, and that its emission is sensitive to solvent. In addition, 7-azatryptophan can be incorporated into synthetic peptides and bacterial protein. In this article, the interactions of 7-azaindole with its environment are discussed. Special attention is directed to the difference in its fluorescence properties in water as opposed to nonaqueous solvents. The sensitivity of 7-azaindole to its environment is demonstrated and then exploited by studying it and its analogs in peptides and in complexes with larger proteins containing many tryptophan residues.

Finding the right pathway: Reaction of isotopomers of ethylene with acetate species (see scheme) adsorbed on a Pd(111) surface in ultrahigh vacuum has shown that vinyl acetate is produced by insertion of ethylene into the acetate species to form an acetoxyethyl intermediate, which decomposes by β‐hydride elimination to yield vinyl acetate.

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L-Enduracididine (L-End) is a nonproteinogenic amino acid found in a number of bioactive peptides, including the antibiotics teixobactin, enduracidin, and mannopeptimycin. The potent activity of these compounds against antibiotic-resistant pathogens like MRSA and their novel mode of action have garnered considerable interest for the development of these peptides into clinically relevant antibiotics. This goal has been hampered, at least in part, by the fact that L-End is difficult to synthesize and not currently commercially available. We have begun to elucidate the biosynthetic pathway of this unusual building block. In mannopeptimycin-producing strains, like Streptomyces wadayamensis, L-End is produced from L-Arg by the action of three enzymes: MppP, MppQ, and MppR. Herein, we report the structural and functional characterization of MppP. This pyridoxal 5'-phosphate (PLP)-dependent enzyme was predicted to be a fold type I aminotransferase on the basis of sequence analysis. We show that MppP is actually the first example of a PLP-dependent hydroxylase that catalyzes a reaction of L-Arg with dioxygen to yield a mixture of 2-oxo-4-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid in a 1.7:1 ratio. The structure of MppP with PLP bound to the catalytic lysine residue (Lys221) shows that, while the tertiary structure is very similar to those of the well-studied aminotransferases, there are differences in the arrangement of active site residues around the cofactor that likely account for the unusual activity of this enzyme. The structure of MppP with the substrate analogue D-Arg bound shows how the enzyme binds its substrate and indicates why D-Arg is not a substrate. On the basis of this work and previous work with MppR, we propose a plausible biosynthetic scheme for L-End.

The non-proteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens including methicillin-resistant Staphylococcus aureus. Enduracididine is produced in Streptomyces hygroscopicus by three enzymes, MppP, MppQ, and MppR. Based on primary sequence analysis, MppP and Q are pyridoxal-5'-phosphate-dependent aminotransferases; MppR shares low, but significant, sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme, and the intermediates involved in the route to enduracididine are currently unknown. Herein we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of a mixture of "4-hydroxy-2-ketoarginine" and "2-ketoenduracididine" shows only the correct 4R-enantiomer of "2-ketoenduracididine" bound to the enzyme. These data, together with bioinformatic analysis of MppR homologs, identifies a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function.