Supplementary Information

Substrate Specificity of Benzamide Synthetase Involved in 4-Hydroxy-3-Nitrosobenzamide Biosynthesis

Akio Noguchi, Sueharu Horinouchi#, and Yasuo Ohnishi*

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo.

* Correspondence: Yasuo Ohnishi, Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail:

# Deceased on 12th July 2009

Supplementary Figure S1. Phylogenetic analysis of Gn-ATs. The sequences used for alignment were those of Gn-ATs involved in secondary metabolite synthetic pathways in Streptomyces and other bacteria, as well as the asparagine synthetases from bacteria and mammals (see Table S1). An unrooted tree was constructed by the CLUSTALW multiple alignment program using the following settings: MATRIX blosum, GAPOPEN 10.0, GAPEXT 0.2, GAPDIST 8, MAXDIV 40, bootstrap counts 1000.1 Numbers indicate bootstrap values > 800. Bar = 0.1 amino acid substitutions per site. Among these phylogenetically related bacterial Gn-ATs, NspN,2 OxyD,3 SsfD,4 PcsA,5 PcsB,6 TsrC,7 PhzH-Pa,5 and PhzH-Pc6 had been evaluated for their function in secondary metabolite syntheses in vivo. Only NspN2 and PcsB6 had also been characterized in vitro. In contrast, ORF5,8 SioC, ORF1,9 SfaP, TcsG, FdmV,10 SanV,11 and LlpA12 had not yet been fully characterized. Their involvement in secondary metabolism has been suggested because they are encoded in biosynthetic gene clusters responsible for their respective compounds. GrhP,13 RubR, ORF11,14 and TblS, which belong to the same clade, have no catalytic Cys residue, which is characteristic of the class II glutamine amidotransferase domain. This indicates that these proteins have lost their Gn-AT activity. Apart from the clade including NspN, ORF71x8,15 MoeF5,16 Ste10,17 ORF3, ORF4, ORF16,18 ORF18,19 and ORF2120 also seem to be involved in secondary metabolism processes, however these Gn-ATs have not yet been fully characterized. Asparagine synthetases (AsnB-Ec, AsnB-Vc, ASNS and AS), which catalyze the conversion of aspartic acid to asparagine, are phylogenetically distant from NspN.


Supplementary Table S1. Gn-AT enzymes used for phylogenetic analysis.

a Amino acid sequence identity to NspN.

b These proteins have no catalytic Cys, indicating a loss of Gn-AT activity.


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