The plant enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138. Determination of the kinetic constants of the overexpressed and purified enzymes revealed that mutagenesis led in each case to diminished activity. Mutants S203A, R354A and Y351F showed a decrease in k cat by factors of 435, 130 and 235, respectively. Mutants F400A, Q488A and L138H showed a 345-, 615-and 14-fold lower k cat , respectively. The greatest loss of activity occurred in the PAL mutants N260A, Q348A and Y110F, which were 2700, 2370 and 75 000 times less active than wild-type PAL. To elucidate the possible function of the mutated amino-acid residues in PAL we built a homology model of PAL based on structural data of HAL and mutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowed us to propose possible roles for the corresponding residues in PAL catalysis.

Radical SAM enzymes have only recently been recognized as an ancient family sharing an unusual radical-based reaction mechanism. This late appreciation is due to the extreme oxygen sensitivity of most radical SAM enzymes, making their characterization particularly arduous. Nevertheless, realization that the novel apposition of the established cofactors S-adenosylmethionine and [4Fe-4S] cluster creates an explosive source of catalytic radicals, the appreciation of the sheer size of this previously neglected family, and the rapid succession of three successfully solved crystal structures within a year have ensured that this family has belatedly been noted. In this review, we report the characterization of two enzymes: the established radical SAM enzyme, HemN or oxygen-independent coproporphyrinogen III oxidase from Escherichia coli, and littorine mutase, a presumed radical SAM enzyme, responsible for the conversion of littorine to hyoscyamine in plants. The enzymes are compared to other radical SAM enzymes and in particular the three reported crystal structures from this family, HemN, biotin synthase and MoaA, are discussed.

Synthesis of 1-Substituted 5-Aminoimidazole-4-carbaldehydes and 8-Amino-1-(2-fluorobenzyl)imidazo[4',5':5,6]pyrido[2,3-d]pyrimidine S y n t h e s i s o f 1 -S u b s t i t u t e d 5 -A m i n o i m i d a z o l e -4 -c a r b a l d e h y d e sAbstract: The syntheses of a number of 1-substituted 5-aminoimidazole-4-carbaldehydes 3, by the reduction of the corresponding 5amino-4-cyanoimidazole derivatives is reported using Li-AlH(OEt) 3 , prepared in situ from LiAlH 4 and ethyl acetate. The compounds of type 1 are useful intermediates for the synthesis of elongated adenine derivatives.Although a variety of conditions have been reported 1-3 for the reduction of heterocyclic nitriles to aldehydes, to our knowledge, the only reported synthesis of 5-aminoimidazole-4-carbaldehydes 3 (R 1 = R 2 = Me; R 1 = Me, R 2 = Ph) is by the catalytic hydrogenation of the corresponding 4aminoimidazole-5-carbonitriles using 10% Pd/C in dilute sulfuric acid. 4 These compounds have been shown to be useful reagents for the synthesis of imidazo[4,5-b]pyridine derivatives. 4 B. L. Booth et al.

Synthesis of 1-Substituted 5-Aminoimidazole-4-carbaldehydes and 8-Amino-1-(2-fluorobenzyl)imidazo [4',5':5,6]pyrido[2,3-d]pyrimidine. -Various title compounds (III) are prepared as shown. The synthetic utility of the products is proved by transformation of (IIIe) to the pharmaceutically interesting ring system (VIII). -(BOOTH, BRIAN L.; CARPENTER, ROBERT A.; MORLOCK, GABY; MAHMOOD, ZAHID; PRITCHARD, ROBIN B.; Synthesis (2001) 16, 2393-2396; Dep. Chem., Univ. Manchester Inst. Sci. Technol., Manchester M60 1QD, UK; EN)