Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (l-alanine-[2,3-epoxycyclohexano-4]-l-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in B. subtilis. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of l-anticapsin. These data, however, were not consistent with the putative functional annotation for these proteins whereby BacA, a prephenate dehydratase along with a potential isomerase/guanylyl transferase, BacB and an oxidoreductase, BacC, could synthesize l-anticapsin. Here we demonstrate that BacA is a decarboxylase that acts on prephenate. Further, based on the biochemical characterization and the crystal structure of BacB, we show that BacB is an oxidase that catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to l-anticapsin. This protein is a bi-cupin, with two putative active sites each containing a bound metal ion. Additional electron density at the active site of the C-terminal domain of BacB could be interpreted as a bound phenylpyruvic acid. A significant decrease in the catalytic activity of a point variant of BacB with a mutation at the N-terminal domain suggests that the N-terminal cupin domain is involved in catalysis.

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The unique multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been exploited to generate polypeptide fragments which, when cloned and expressed, reassemble in the presence of cofactors to yield a catalytically competent enzyme. Multidimensional multinuclear NMR methods have been employed for obtaining near complete sequence specific NMR assignments for backbone HN, 15N, 13Calpha and 13Cbeta atoms of the FAD binding domain of ilvB on samples that were isotopically enriched in 2H, 13C and 15N. Unambiguous assignments were obtained for 169 of 177 backbone Calpha atoms and 127 of 164 side chain Cbeta atoms. The secondary structure determined on the basis of observed 13Calpha secondary chemical shifts and sequential NOEs agrees well with the structure of this domain in the catalytic subunit of yeast AHAS. Binding of ilvN to the ilvBalpha and ilvBbeta domains was studied by both circular dichroism and isotope edited solution nuclear magnetic resonance methods. Changes in CD spectra indicate that ilvN interacts with ilvBalpha and ilvBbeta domains of the catalytic subunit and not with the ilvBgamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvBbeta and proximal to the intrasubunit ilvBalpha/ilvBbeta domain interface. The implication of this interaction on the role of the regulatory subunit on the activity of the holoenzyme is discussed.

While it is well known that introduction of Pro residues into the interior of protein alpha-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein alpha-helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 A. In wild-type thioredoxin, P40 is located in the interior of helix two, a long alpha-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin.

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