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The Structure of Pyrococcus Furiosus Glutamate Dehydrogenase Reveals

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The Structure of Pyrococcus Furiosus Glutamate Dehydrogenase Reveals View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures KSP Yip', TJ Stillman', KL Britton1 , PJ Artymiuk', PJ Baker'1, SE Sedelnikova1, PC Engel' t, A Pasquo2 , R Chiaraluce2 , V Consalvi2, R Scandurra2 and DW Rice1* 1 The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, PO Box 594, Sheffield S10 2UH, UK and 2 Dipartimento di Scienze Biochimiche, Universita La Sapienza, Roma 00185, Italy Background: The hyperthermophile Pyrococcusfuriosus is glutamate dehydrogenase from the mesophile Clostridium one of the most thermostable organisms known, with symbiosum. an optimum growth temperature of 1000C. The proteins Conclusions: Comparison of the structures of these two from this organism display extreme thermostability. We enzymes has revealed one major difference: the structure have undertaken the structure determination of glutamate of the hyperthermophilic enzyme contains a striking series dehydrogenase from P. furiosus in order to gain further of ion-pair networks on the surface of the protein insights into the relationship between molecular structure subunits and buried at both interdomain and inter- and thermal stability. subunit interfaces. We propose that the formation of such Results: The structure of P. furiosus glutamate dehydro- extended networks may represent a major stabilizing genase, a homohexameric enzyme, has been determined feature associated with the adaptation of enzymes to at 2.2 A resolution and compared with the structure of extreme temperatures. Structure 15 November 1995, 3:1147-1158 Key words: glutamate dehydrogenase, hyperthermophile, Pyrococcus furiosus, thermal stability, X-ray crystallography Introduction metabolism and which catalyze the oxidative deamina- The Archaea represent a phylogenetically distinct evo- tion of L-glutamate to 2-oxoglutarate and ammonia lutionary domain whose members possess various extra- with the concomitant reduction of one molecule of ordinary properties, which include the ability of some NAD(P)+ . Biochemical studies on the GluDHs from a species to grow at temperatures in excess of 100 0 C. diverse range of sources have shown that the vast major- Common habitats of these remarkable microorganisms ity of these enzymes are based on a hexamer of identical are submarine hydrothermal areas and continental solfa- subunits [8]. Analysis of the amino acid sequence for Pf taric fields [1-3]. Not surprisingly, the proteins from GluDH has shown that it displays considerable similarity these organisms display extreme thermostability and the to other members of this enzyme family [12,13]. In analysis of their structures is expected to provide insights particular, Pf GluDH shows 34% sequence identity with into the molecular mechanisms which Nature employs the GluDH from the mesophile Clostridium symbiosum in order to achieve this. Understanding the basis of (Cs) [14], a molecule of known three-dimensional this thermostability may allow us to engineer proteins to structure [15,16], which has a half-life at 520 C of withstand not only high temperatures but also environ- approximately 20 min [17]. The striking difference in ments even more hostile to life but relevant to the thermostability combined with the sequence similarity conditions used in many industrial processes [4]. between these two enzymes encouraged us to solve the structure of Pf GluDH. In this paper we describe the The archaeon Pyrococcusfuriosus (Pf), is one of the most structure determination of this enzyme and the results thermostable organisms known, with an optimum growth of the comparative analysis with the structure of the temperature of 100°C [5]. The glutamate dehydrogenase GluDH from Cs. This has allowed us to highlight struc- (GluDH) from this organism accounts for up to 20% of tural features of the Pf enzyme which may contribute to the total cell protein and is extremely thermostable, with its superior thermal properties. a half-life at 1000C of 12 h [6,7]. The abundance of GluDH, coupled with the wealth of biochemical data on this enzyme from a diverse range of mesophilic organisms Results and discussion [8], has created considerable interest in it as a model Description of the Pyrococcus furiosus GluDH structure system for the analysis of thermostability [9-11]. Overall, the fold of the GluDH from Pf is very similar to that of the hexameric GluDH from Cs GluDHs are ubiquitous enzymes which occupy an [15,16], enabling us to undertake a detailed comparison important branch point between carbon and nitrogen of their molecular structures. The secondary-structure *Corresponding author. tPresent address: Department of Biochemistry, University of Dublin, Belfield, Dublin 4, Eire. © Current Biology Ltd ISSN 0969-2126 1147 1148 Structure 1995, Vol 3 No 11 assignments and structure-based sequence alignment for In previous studies, a decrease in the lengths of the loops Pf and Cs GluDHs are given in Figure 1. A schematic connecting elements of regular secondary structure has representation of the structure of a single subunit of Pf been suggested to be important for thermostability [18]. GluDH is shown in Figure 2. The structure of a single Comparisons between the two GluDHs show that there subunit of this enzyme consists of two domains sepa- are only two regions of major structural difference. rated by a deep cleft. Domain I is largely composed of Firstly, at the N terminus, the Pf GluDH is some 21 residues from the N-terminal portion of the polypep- residues shorter, corresponding to the loss of helix al in tide chain and is responsible for directing the self- Cs GluDH (15 residues) and the replacement of two assembly of the subunits into a hexamer with 32 short helices, a3 and o4, by a loop. The structural symmetry, whereas domain II contains the nucleotide- rearrangement caused by these changes does indeed lead binding site. The domain interface is dominated by to a more compact character for Pf GluDH in this region interactions involving four long x helices (a6, a12, a13 (Fig. 3a). However, the sequence at the N terminus of and ot14 in Pf GluDH). The active-site pocket is located GluDH is the most variable region across the enzyme in the cleft region and contains residues provided by family. Helix axl is also deleted from the structure of both domains. A description of the structure of the GluDH from the mesophilic organism Neurospora crassa hyperthermophilic Pf GluDH is presented below and is (TJS, DWR, et al., unpublished data) and its absence in compared with Cs GluDH. many other mesophilic species, such as Saccharomyces cere- visiae, is implied from sequence alignment of GluDHs Analysis of secondary structure, hydrogen bonding and loops [14,19,20]. It would therefore appear that this difference The overall similarity between the two enzymes is illus- is not necessarily related to thermostability. Furthermore, trated in Figure 1 and Table 1. The percentage of resi- the shortening of sequences at the N terminus has been dues in helical -or -strand conformations is virtually noted elsewhere and has led to the suggestion that this identical. Furthermore, it is clear from Table 1 that the feature may merely reflect phylogenetic differences [18]. number of main-chain hydrogen bonds, and the average number of protein-protein hydrogen bonds per residue, A second region of significant structural and sequence are very similar and thus the differences between these difference occurs between strands 13i and 3k (residues enzymes in these respects are also very subtle. 267-316 in the Cs enzyme), as shown in Figure 3b. In GDH(CS) 1 kydrvia.vekkyadEPEFVQtvvls..lgpvvdahpy..vALLMZPVIPRvEFRVP ZDDNGKVHVNTGYRVQFN82 GDH(Pf) 1 veQDPYEIvikqleraaqy ------ ieEALEFLPQRIVEVTIPV NDiDGSVKVFTGFRVQHN61 GDH(Cs) 83 GAIGPYKGGLRFAPSVNLSIMKFLGFEQAIKDSLTLPMGGAKGGSOFDPNGKSDREVMRFCQAPIFTELYRHIGPDIDVPAG164 GDH(Pf) 62 WARGPTKGGIRWHPEETLSTVALAA AVMDLPYGKGG IVDPLSDp RLAR YIRAIYDVISYEDIPAP 143 GDH(Cs ) 165 DLGGAREI GYMYGQYRKIVGGf-- yNGVLTGKARSFGGSLVRPEATGYGSVYYVEAVMID -tLVGKTVALAGGNVAW 243 GDH(Pf) 144 DVYTNPQIKAWEDEYETISRRktpaFGIITGK LSIGGSLGRIEATARGASYTIREAAXV ItLKGKTIAIQGYGNAGY 225 GDH(C) 244 GAAKLAE-LGAKAVTLSGPYGYIYDPEGittekiylUasLqrkvqdydkfgqVQFFpg POWIQVDXInPCATQN 324 GDH(Pf) 226 YLATIMSEdFGM VAVSDSKGGIYNP1 ----.dvYkWkneh---- vkWdfgATNIt.. .LLeLVDVILAPAAIZZ 299 Fig. 1. Alignment of the sequences for Clostridium symbiosum (Cs) and Pyro- coccus furiosus (Pf) GluDH based on the superposition of the respective Ca coordinates of each domain. Equivalent residues whose relative positions differ GDH(Cs) 325 DVDLZQAIV VKYIVAnTYYI EVA QNWAPSKA GVLVSGFUMSQNSI IrL8WTAEESVDMNHq 406 GDH(Pf) 300 VITI0XADNI ---kaKIWVANGPVTPEADEILFEK-qILIPDFLNAGGVTVSyFELQNIX CTGYY6IEEVRULD 377 by greater than 2 A are indicated by the use of lower-case letters. The secondary dr1 structural elements of the three-dimen- sional structure of the two GluDHs are shown with helices represented as cylinders and strands shown as arrows. GDH(Cs) 407 TDIHDGSAA..ryggynlVAANIVGFQKIADAO Gi-W 449 The residue numbers for the individual CDH(Pf) 378 TXAFYDVYNiakkni--h.RDAAYVVAVQRVYQAILaDRvkh 419 sequences are given at the beginning and end of each line. Positions of del- otirnc ,ro inrlirtrrl hv, - _LV.IU~I~_ 1 ................. ( ._1C .. i P. furiosus glutamate dehydrogenase Yip et a. 1149 Fig. 2. Stereo
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