research papers 1212 doi:10.1107/S1399004714002016 Acta Cryst. (2014). D70, 1212–1223 Acta Crystallographica Section D Biological Crystallography ISSN 1399-0047 Structural basis of a novel activity of bacterial 6-pyruvoyltetrahydropterin synthase homologues distinct from mammalian 6-pyruvoyltetrahydro- pterin synthase activity Kyung Hye Seo, a ‡ Ningning Zhuang, a Young Shik Park, b,c Ki Hun Park d and Kon Ho Lee a,e * a Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Gyeongsang National University, Jinju 660-701, Republic of Korea, b School of Biological Sciences, Inje University, Kimhae 621-749, Republic of Korea, c Department of Health Science and Technology, Graduate School, Inje University, Kimhae 621-749, Republic of Korea, d Division of Applied Life Science (BK21 Plus), IALS, Gyeongsang National University, Jinju 660-701, Republic of Korea, and e Department of Microbiology, School of Medicine, Gyeongsang National University, Jinju 660-751, Republic of Korea ‡ Present address: Department of Functional Crops, National Institute of Crop Science, Rural Development Administration (RDA), Miryang 627-803, Republic of Korea. Correspondence e-mail: lkh@gnu.ac.kr # 2014 International Union of Crystallography Escherichia coli 6-carboxytetrahydropterin synthase (eCTPS), a homologue of 6-pyruvoyltetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterin in vitro compared with genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin in vivo. Crystal structures of wild-type apo eCTPS and of a Cys27Ala mutant eCTPS complexed with sepiapterin have been determined to 2.3 and 2.5 A ˚ resolution, respectively. The structures are highly conserved at the active site and the Zn 2+ binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologues revealed that two specific residues, Trp51 and Phe55, that are not found in mammalian PTPS keep the substrate bound by stacking it with their side chains. Replacement of these two residues by site- directed mutagenesis to the residues Met and Leu, which are only found in mammalian PTPS, converted eCTPS to the mammalian PTPS activity. These studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and in the specific enzyme activity that differs from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologues. Received 16 October 2013 Accepted 28 January 2014 PDB references: 6-carboxy- tetrahydropterin synthase, 3qn9; 3qn0; C27A mutant, complex with sepiapterin, 3qna 1. Introduction 6-Pyruvoyltetrahydropterin synthase (PTPS; EC 4.2.3.12) is the second enzyme in the biosynthesis of tetrahydrobiopterin (BH 4 ), catalyzing the conversion of dihydroneopterin tri- phosphate (H 2 NTP) to 6-pyruvoyltetrahydropterin (PPH 4 ) in mammals (Tho ¨ny et al. , 2000; Nagatsu & Ichinose, 1999). BH 4 is a well known essential cofactor for aromatic amino-acid hydroxylases and nitric oxide synthases, which play vital roles in the biosynthesis of dopamine and serotonin in higher animals (Tho ¨ ny et al. , 2000; Nagatsu & Ichinose, 1999). BH 4 is essential for various processes and is probably present in every cell or tissue of higher organisms. However, BH 4 is not common in bacteria, although its glycosidic forms have been found in particular species such as cyanobacteria (Chung et al., 2000; Wachi et al., 1995) and the anaerobic photosynthetic bacterial species from the genus Chlorobium (Cho et al. , 1998; Kang et al., 1998). A large number of PTPS homologues have been found in the genomes of many bacteria that are not known to produce BH 4 or its glycosides. These bacterial PTPS homologues can be classified into three groups depending on their activities: bacterial PTPS-I (bPTPS-I), bacterial PTPS-II (bPTPS-II) and bacterial PTPS-III (bPTPS-III) (Kong et al., 2006; Pribat et al.,