The structure of putative N-acetyl glutamate kinase from Thermus thermophilus reveals an intermediate active site conformation of the enzyme Ramya Sundaresan a , Preethi Ragunathan a , Seiki Kuramitsu b,c , Shigeyuki Yokoyama d,e , Thirumananseri Kumarevel b,⇑ , Karthe Ponnuraj a,⇑ a Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India b RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan c Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan d RIKEN Systems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan e Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan article info Article history: Received 8 March 2012 Available online 20 March 2012 Keywords: N-acetyl-L-glutamate kinase Arginine biosynthesis Crystal structure Intermediate conformation Catalytic cycle abstract The de novo biosynthesis of arginine in microorganisms and plants is accomplished via several enzymatic steps. The enzyme N-acetyl glutamate kinase (NAGK) catalyzes the phosphorylation of the c-COO À group of N-acetyl-L-glutamate (NAG) by adenosine triphosphate (ATP) which is the second rate limiting step in arginine biosynthesis pathway. Here we report the crystal structure of putative N-acetyl glutamate kinase (NAGK) from Thermus thermophilus HB8 (TtNAGK) determined at 1.92 Å resolution. The structural analy- sis of TtNAGK suggests that the dimeric quaternary state of the enzyme and arginine insensitive nature are similar to mesophilic Escherichia coli NAGK. These features are significantly different from its thermo- philic homolog Thermatoga maritima NAGK which is hexameric and arginine-sensitive. TtNAGK is devoid of its substrates but contains two sulfates at the active site. Very interestingly the active site of the enzyme adopts a conformation which is not completely open or closed and likely represents an interme- diate stage in the catalytic cycle unlike its structural homologs, which all exist either in the open or closed conformation. Engineering arginine biosynthesis pathway enzymes for the production of L-arginine is an important industrial application. The structural comparison of TtNAGK with EcNAGK revealed the struc- tural basis of thermostability of TtNAGK and this information could be very useful to generate mutants of NAGK with increased overall stability. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Arginine biosynthesis in microorganisms, plants (also unicellu- lar algae) and fungi is normally carried out from L-glutamate to arginine via n-acetylated intermediates in eight enzymatic steps (Supplementary Fig. S1) [1–5]. Microorganisms follow either a lin- ear [1] or cyclic pathway [1,4–5] based on the formation of the intermediate product L-ornithine catalyzed by acetyl ornithinase (AO) or ornithine acetyl transferase (OAT), respectively [5]. Orni- thine is later converted to arginine via citrulline and argininosucci- nate [5,6]. Microorganisms like Escherichia coli and Sulfolobus solfotaricus follow a linear pathway while Thermatoga maritima and Thermus aquaticus follow a more evolved cyclic pathway. Ther- mus thermophilus, an extreme thermophile is believed to take the cyclic route for ornithine production like T. maritima [7]. N-acetyl glutamate kinase (NAGK), a key player in second step of arginine biosynthesis catalyzes the phosphorylation of the c- COO À group of N-acetyl-L-glutamate (NAG) by adenosine triphos- phate (ATP) and an ideal candidate for feedback inhibition by argi- nine in the cyclic pathway. Two classes of NAGKs have evolved based on their sensitivity to arginine namely, arginine-sensitive and arginine-insensitive forms [2,4]. Some of arginine-sensitive NAGKs structurally well characterized till date are from Pseudomo- nas aeruginosa (PaNAGK) [8], T. maritima (TmNAGK) [8], Arabidopsis thaliana (AtNAGK) [7] and Synecococcus elongatus (SeNAGK) [9], while the only arginine insensitive NAGK crystallized and analyzed till date is from E. coli (EcNAGK) [2]. EcNAGK is a homodimer while arginine-sensitive forms are hexamers. Here we report the crystal structure of NAGK from T. thermophilus (TtNAGK) at a resolution of 1.92 Å. TtNAGK is a dimer unlike its thermophilic homolog (TmNAGK), making it the first NAGK of thermophilic origin pos- sessing dimeric structure. The absence of NAGK in mammals [10,11] which do not take up this acetylated route for arginine synthesis makes it an attractive target for the development of new antimicrobials for inhibiting 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2012.03.072 ⇑ Corresponding authors. Fax: +91 44 2220 0122 (K. Ponnuraj), fax: +81 791 58 2917 (T. Kumarevel). E-mail addresses: tskvel@spring8.or.jp (T. Kumarevel), karthe@unom.ac.in, pkarthe@hotmail.com (K. Ponnuraj). Biochemical and Biophysical Research Communications 420 (2012) 692–697 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc