Pantoea agglomerans phenylalanine aminomutase (PaPAM) is an enzyme that reacts with (2R)-&alpha-phenylalanine to produce (3S)-&beta-phenylalanine in the biosynthetic production of the antibiotic Andrimid. The mechanism by which this class of enzymes achieves this transformation is debated. The crystal structure of the (3S)-&beta-phenylalanine bound PaPAM was determined with both (2R)-&alpha-phenylalanine and (3S)-&beta-phenylalanine bound to the active site providing evidence that this class of enzymes utilizes an amino-group alkylation pathway.Benzoate Coenzyme A (CoA) Ligase from Rhodopseudomanas palustris (BadA) catalyzes the ligation of Coenzyme A to a variety of benzoic acids in the presence of adenosine triphosphate (ATP). Benzyl-CoAs are useful in the biosynthesis of small molecules. The crystal structure was therefore determined with various natural and unnatural substrates bound to the active site and within water exposed channels. These structures demonstrate the mode by which this enzyme catalyses the reaction; aiding in our understanding of its mode of action and our ability to increase the promiscuity of the enzyme to produce benzyl-CoA derivatives. Small Nuclear RNA Activating Protein (SNAPc) is a human nuclear transcription factor composed of five subunits that activates the transcription of small nuclear RNA by recruiting RNA polymerase I, II or III to various promoters. Although different polymerases are recruited, it is the presence of the same element, the proximal sequence element (PSE), upstream of the transcription start site that recruits SNAPc. The U1 promoter contains such an element to which SNAPc recruits RNA polymerase II (Pol II). In the case of the U6 promoter, the presence of a TATA box additionally recruits the TATA Binding Protein (TBP) and ultimately RNA Polymerase III (Pol III) is activated. However there is no evidence that it is the presence of TBP that would discriminate between Polymerase II and Polymerase III recruitment. To understand this phenomenon, a truncated version of SNAPc was co-expressed in E. coli for crystallization studies. Though a complex of the four proteins could be produced, attempts to crystallize it were unsuccessful. Transcription factor IIIB (TFIIIB) recruits Pol III in budding yeast such as Saccharomyces cerevisiae ( Sc ). In gene internal promoters containing an A Box element, Transcription Factor IIIA (TFIIIA) is first recruited which in turn recruits TFIIIC which then recruits TFIIIB. The gene internal promoters such as those for the tRNA containing an A Box element recruit TFIIIC directly, which in turn recruits TFIIIB. In the case of the U6 snRNA promoter which contains a TATA Box element it was shown that TFIIIB is recruited directly to the promoter via the subunit TBP. In order to facilitate crystallization, a BRF1-TBP triple fusion was generated containing the amino and carboxyl termini of Brf1 with TBP inserted in between. This triple fusion was shown to have the same activity as the separate units; however it proved to be unsuitable for crystallization.
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