1. Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to alpha-carbon cross-links: formation and reduction of alpha-thio-alpha-amino acid derivatives
Karen E Kawulka, Tara Sprules, Christopher M Diaper, Randy M Whittal, Ryan T McKay, Pascal Mercier, Peter Zuber, John C Vederas Biochemistry. 2004 Mar 30;43(12):3385-95. doi: 10.1021/bi0359527.
The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled [(13)C,(15)N]medium derived from Anabaena sp. grown on sodium [(13)C]bicarbonate and [(15)N]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-(13)C,(15)N]-L-phenylalanine and [U-(13)C,(15)N]-L-threonine using otherwise unlabeled media. The results demonstrate that in addition to having a cyclized peptide backbone (amide between N and C termini), three cross-links are formed between the sulfurs of Cys13, Cys7, and Cys4 and the alpha-positions of Phe22, Thr28, and Phe31, respectively. The stereochemistry of all residues in 1 except for the three modified ones was confirmed to be L by complete desulfurization with nickel boride, acid hydrolysis to the constituent amino acids, and conversion of these to the corresponding pentafluoropropanamide isopropyl esters for chiral GC MS analysis. The stereochemistry at the modified residues was determined by subjecting each of the eight possible stereoisomers of 1 to eight rounds of ARIA structure calculations, starting with the same NMR peak files and assignments. The stereoisomer with the l stereochemistry at Phe22 (alpha-R) and d stereochemistry at Thr28 (alpha-S) and Phe31 (alpha-S) (LDD isomer) fit the NMR data, giving the lowest energy family of structures with the best rmsd. Thus, biochemical formation of the unusual thio links proceeds with net retention of configuration at Phe22, and inversion at Thr28 and Phe31. Model amino acid derivatives bearing a sulfide moiety at the alpha-carbon were synthesized by reaction of the corresponding alpha-alkoxy compounds with benzyl thiol and SnCl(4). Separation of their pure stereoisomers and desulfurization with nickel boride demonstrated that the reduction of such compounds proceeds with epimerization, in contrast to the previously reported retention of stereochemistry for analogous reaction of steroidal sulfides. However, desulfurization of subtilosin A to cyclic peptide 14, which is inactive as an antimicrobial agent, occurs with inversion of stereochemistry at the alpha-carbons of Phe22 and Thr28 and with 4:1 retention at Phe31. This indicates that the desulfurization reaction proceeds via an N-acyl imine and that the structure of the surrounding peptide controls the geometry of reduction. Posttranslational linkage of a thiol to the alpha-carbon of an amino acid residue is unprecedented in ribosomally synthesized peptides or proteins, and very rare in secondary metabolites. Subtilosin A (1) represents a new class of bacteriocins.
2. Structures of D-amino-acid amidase complexed with L-phenylalanine and with L-phenylalanine amide: insight into the D-stereospecificity of D-amino-acid amidase from Ochrobactrum anthropi SV3
Seiji Okazaki, Atsuo Suzuki, Tsunehiro Mizushima, Hidenobu Komeda, Yasuhisa Asano, Takashi Yamane Acta Crystallogr D Biol Crystallogr. 2008 Mar;64(Pt 3):331-4. doi: 10.1107/S0907444907067479. Epub 2008 Feb 20.
The crystal structures of D-amino-acid amidase (DAA) from Ochrobactrum anthropi SV3 in complex with L-phenylalanine and with L-phenylalanine amide were determined at 2.3 and 2.2 A resolution, respectively. Comparison of the L-phenylalanine amide complex with the D-phenylalanine complex reveals that the D-stereospecificity of DAA might be achieved as a consequence of three structural factors: (i) the hydrophobic cavity in the region in which the hydrophobic side chain of the substrate is held, (ii) the spatial arrangement of Gln310 O and Glu114 O epsilon2 that fixes the amino N atom of the substrate and (iii) the existence of two cavities that keep the carboxyl/amide group of the substrate near or apart from Ser60 O gamma.
3. Discovery of a solid solution of enantiomers in a racemate-forming system by seeding
Jun Huang, Shuang Chen, Ilia A Guzei, Lian Yu J Am Chem Soc. 2006 Sep 13;128(36):11985-92. doi: 10.1021/ja063450l.
A racemic liquid of opposite enantiomers usually crystallizes as a racemic compound (racemate), rarely as a conglomerate, and even more rarely as a solid solution. We discovered a Type II solid solution (mixed crystal) of the enantiomers of the chiral drug tazofelone (TZF) by seeding its racemic liquid with enantiomerically pure crystals (enantiomorphs). Without seeding, the racemic liquid crystallized as a racemic compound. The crystal structure of this solid solution resembles that of the enantiomorph but has static disorder arising from the random substitution of enantiomers. This solid solution is a kinetic product of crystallization made possible by its faster growth rate compared to that of the competing racemate (by 4- to 40-fold between 80 and 146 degrees C). The free energy of the solid solution continuously varies with the enantiomeric composition between those of the conglomerate and the racemates. The existence of the TZF solid solution explains the absence of eutectic melting between crystals of different enantiomeric compositions. The ability of TZF to simultaneously form racemate and solid solution originates from its conformational flexibility. Similar solid solutions of enantiomers may exist in other systems and may be discovered in similar ways. The study demonstrates the use of cross-nucleation for discovering and engineering crystalline materials to optimize physical properties.
