Substrate specificity of alkaline serine proteinase isolated from photosynthetic bacterium, Rubrivivax gelatinosus KDDS1 q Somporn Tanskul, a Kohei Oda, a, * Hiroshi Oyama, a Napavarn Noparatnaraporn, b Masahiko Tsunemi, c and Katsumi Takada c a Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan b Department of Microbiology, Kasetsart University, Jatujak, Bangkok 10903, Thailand c Peptide Institute, Inc., Minoh, Osaka 562-8686, Japan Received 6 August 2003 Abstract A novel type of fluorescence resonance energy transfer (FRET) combinatorial libraries were used for the characterization of alkaline serine proteinase produced from Rubrivivax gelatinosus KDDS1. This enzyme was the first serine proteinase charac- terized from photosynthetic bacteria. The proteinase was found to prefer Met and Phe at the P 1 position, Ile and Lys at the P 2 position, and Arg and Phe at the P 3 position. To date, no serine proteinase has exhibited a preference for Met at the P 1 position. Thus, the alkaline serine proteinase from R. gelatinosus KDDS1 is very unique in terms of substrate specificity. A highly sensitive substrate, Boc-Arg-Ile-Met-MCA, was synthesized for kinetic study based on the results reported here. The optimum pH of the enzyme for this substrate was pH 10.7, and the values of k cat , K m , and k cat /K m were 23.7 s 1 , 15.4 lM, and 1.54 lM 1 s 1 , respectively. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Fluorescence resonance energy transfer; Combinatorial library; Serine proteinase; Photosynthetic bacteria; Kinetic parameters We have recently succeeded in isolating a photosyn- thetic bacterium KDDS1 strain, identified as Rubrivivax gelatinosus that can secrete proteinase extracellularly. 1 The secreted enzyme was characterized as an alkaline serine proteinase, the first one characterized from pho- tosynthetic bacteria. In order to explore the active site cleft of the enzyme, analysis of the substrate specificity was carried out using a novel type of FRET combinatorial libraries. Materials and methods Analysis of substrate specificity using FRETS-25Xaa-libraries 1. FRETS-25Xaa-libraries. FRETS-25Xaa contains a highly fluo- rescent 2-(N-methylamino)benzoyl (Nma) group linked to the side chain of the amino-terminal D-2,3-diamino propionic acid (D-A2pr) residue, which is efficiently quenched by a 2,4-dinitrophenyl (Dnp) group linked to the e-amino function of Lys as shown below. Xaa represents the fixed position where each of the 19 natural amino acids excluding Cys was incorporated. A mixture of 5 amino acid residues (P, Y, K, I, and D) was incorporated at the Yaa position along with a mixture of 5 amino acid residues (F, A, V, E, and R) at the Zaa position for each fixed Xaa. This provides a peptide mixture of 25 combinations of each Xaa series resulting in a combinatorial library with a total of 475 peptide substrates in 19 separate pools. When an Biochemical and Biophysical Research Communications 309 (2003) 547–551 www.elsevier.com/locate/ybbrc BBRC q Abbreviations: FRETS, fluorescence resonance energy transfer substrate; MCA, peptidyl-4-methyl-coumaryl-7-amides; AMC, 7-ami- no-4-methylcoumarin; Nma, 2-(N-methylamino)benzoyl; D-A2pr, D-2,3-diamino propionic acid; Dnp, 2,4-dinitrophenyl. * Corresponding author. Fax: +81-75-724-7760. E-mail address: bika@ipc.kit.ac.jp (K. Oda). 1 K. Oda, S. Tanskul, H. Oyama, N. Noparatnaraporn, Purifica- tion and characterization of alkaline serine proteinase from photosyn- thetic bacterium, R. gelatinosus KDDS1 (2003) (under submission). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.08.035