A Role for Quaternary Structure in the Substrate Specificity of Leucine

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector A role for quaternary structure in the substrate specificity of leucine dehydrogenase Patrick J Baker*t, Andrew P Turnbullt, Svetlana E Sedelnikova, Timothy J Stillman and David W Rice The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, PO Box 594, Sheffield S10 2UH, UK Background: Glutamate, phenylalanine and leucine separated by a deep cleft. The nicotinamide ring of the dehydrogenases catalyze the NAD(P)+-linked oxidative NAD+ cofactor binds deep in this cleft, which is thought deamination of L-amino acids to the corresponding to close during the hydride transfer step of the catalytic 2-oxoacids, and sequence homology between these cycle. enzymes clearly indicates the existence of an enzyme Conclusions: Comparison of the structure of leucine superfamily related by divergent evolution. We have dehydrogenase with a hexameric glutamate dehydroge- undertaken structural studies on a number of members of nase has shown that these two enzymes share a related this family in order to investigate the molecular basis of fold and possess a similar catalytic chemistry. A mecha- their differential amino acid specificity. nism for the basis of the differential amino acid specificity Results: We have solved the X-ray structure of the between these enzymes involves point mutations in the leucine dehydrogenase from Bacillus sphaericus to a resolu- amino acid side-chain specificity pocket and subtle tion of 2.2 A. Each subunit of this octameric enzyme changes in the shape of this pocket caused by the differ- contains 364 amino acids and folds into two domains, ences in quaternary structure. Structure 15 July 1995, 3:693-705 Key words: amino acid dehydrogenase superfamily, leucine dehydrogenase, quaternary structure, substrate specificity Introduction dehydrogenase (GluDH; 20% identical), indicating the The oxidative deamination of amino acids to their corre- existence of an enzyme superfamily with differential sponding keto acids is of central importance in metabo- substrate specificity [4,6]. Homology-based modelling lism and is catalyzed by the family of amino acid studies have shown that when the sequences of LeuDH dehydrogenases. These enzymes also provide an impor- and PheDH are mapped onto the three-dimensional tant route for the incorporation of ammonia into organic structure of GluDH, the secondary-structure elements in compounds and link the metabolism of carbohydrates the core of the two domains of GluDH appear to be and amino acids. Leucine dehydrogenase (LeuDH) (E.C. conserved, and that residues implicated in catalysis are 1.4.1.9) catalyzes the reversible oxidative deamination of preserved [7]. Conversely, a small number of point muta- L-leucine to 2-ketoisocaproate, with the corresponding tions are found in the substrate side-chain-binding reduction of the cofactor NAD+: pocket which are thought to account for the differences in substrate specificity. leucine+NAD++H209 NADH+NH 4++H++2-ketoisocaproate LeuDH was first isolated from Bacillus cereus [1] and has The quaternary structure of LeuDH has been reported as been characterized in many Bacillus species [2], where it hexameric with' subunit molecular weight 41000 Da for functions in the catabolism of branched-chain amino the B. sphaericus strain IF03525 [8]. However, electron acids. The preferred substrate is always leucine. However, microscopy studies indicate that LeuDH from B. cereus is other amino acids, such as valine and isoleucine, are also an octamer with 42 symmetry and a subunit molecular accepted by the enzyme [3]. weight of-40000 Da [9]. The LeuDH from B. sphaericus strain ATCC4525 has been purified, crystallized and The LeuDHs from B. stearothermophilus [4] and Thermo- shown to have a relative molecular mass of 41 000 by actinomyces intermedius [5] have been cloned and polyacrylamide gel electrophoresis [10]. Analysis of a sequenced and contain 429 and 366 amino acids, respec- self-rotation function calculated on a preliminary data set tively. A comparison of their amino acid sequences has shown that the quaternary structure for this enzyme shows that the additional residues in the B. stearo- is that of an octamer arranged in 42 symmetry [10]. thermophilus enzyme form an extension at the C termi- This contrasts with the hexameric structure seen in nus. Sequence alignment studies have revealed the GluDH and is consistent with the location of the major existence of strong sequence similarity between LeuDH insertions and deletions in the aligned sequences of and phenylalanine dehydrogenase (PheDH; 50% identi- GluDH and LeuDH which are found near the threefold cal) and a somewhat more remote similarity to glutamate axis of GluDH [7]. *Corresponding author. tPJB and APT contributed equally to this work. © Current Biology Ltd ISSN 0969-2126 693 694 Structure 1995, Vol 3 No 7 In this paper we report the structure of the LeuDH from type that is the best fit to the observed electron density. B. sphaericus, strain ATCC4525. The structure reveals the At the surface of the protein this protocol must inevitably molecular basis of the substrate specificity, identifies lead to considerable sequence errors, as the electron den- residues lying in the catalytic site, defines the features sity for residues such as glycine, alanine and serine will be responsible for the differences in quaternary structure indistinguishable from weak electron density features between LeuDH and GluDH, and suggests a role for the associated with disordered residues. The sequences of the change in quaternary structure in substrate recognition. LeuDHs from B. stearothermophilus, T intermedius and the final X-ray model sequence of the B. sphaericus enzyme are aligned with the GluDH from Clostridium symbiosum Results in Figure 1. In the final B. sphaericus LeuDH model 57 Structure determination residues are different from those found in either the Crystals of LeuDH belong to space group 4, with a B. stearothermophilus or T intermedius LeuDHs. dimer in the asymmetric unit [10]. As no DNA sequence is yet available for the LeuDH from B. sphaericus, the The LeuDH molecule model has been constructed using a combination of the The LeuDH subunit is constructed from 14 aot-helices sequence for the N-terminal 47 residues, derived from and 12 3-strands, which comprise some 75% of the protein sequencing, together with a consensus sequence polypeptide chain. The secondary-structure assignments from B. stearothermophilus and 7T intermedius LeuDHs. At are given in Figure 1 and a schematic stereo representa- the C terminus there is no sign in the electron-density tion of the structure of a single subunit of LeuDH is map of an extension in the sequence similar to that found shown in Figure 2. The secondary-structure elements in B. stearothermophilus LeuDH, thus we assume that the fold into two domains, separated by a deep cleft. sequence for the B. sphaericus enzyme is of a similar Domain I consists of residues 1-136 and 332-364 and is length to that of the T intermedius enzyme although, at composed of a mixed parallel/antiparallel 3-sheet of six present, we cannot discount the possibility of there being strands (3a-3f), flanked on each side by two aot-helices additional residues at the C terminus which are disor- (aotl,ot2 and ao3,aot4). A short helix (a5) joins the last dered in the electron-density map. In certain places in strand of domain I (f) with the long -helix (6) the model the electron-density map indicates that neither which leads into domain II. This domain (residues the B. stearothermophilus nor the 77Tintermedius sequence is 137-331) resembles the classical dinucleotide-binding correct and in these positions we have selected a residue domain and folds into a central six-stranded parallel <-c(1---> <---a--> <---fb-> <-3c-> <---- -- 2.. LeuDH (X-ray) EIFKYMEKYDYEQLVFCQDEA --SGLKA _68 LeuDH(Bst) MELFKYMTYDYEVLEQDKE ---- SGLKAIIAITTGPAllWLY 68 LeuDH(Ti) MKIFDYMMYDYEQLVMQ SGLKAIICIHVTGPTDE ...--- 68 GluDH(CS) aller vipkVPweddngkvhVN GYRVQFNGAIGYKGAps----VNSIMKFFEQAFK 1141kyvdrviaevekkyadepefvqtveevlsslgpvvda < a1--> <--2-> <-.3-> <-a4-> <-.5> <-sa--> <---b---> <--Pc-> <--- 6--- -> <-pd-> <----a3---- e> <-4----> <pf> <-5-> < -----. 6--> *** ** * * ** . ** * * * LeuDH(X-ray) 69 I G IGDPF- IIQI/--YITAEGTSM EIHQET ------- DYVGISP PVTAYG GSDSL 171 LeuDH(Bst) 69 IGDPR-K EFARFIQG/--(RITAEDVGTVAMDIIYQET--------DYVTGISPEFSGNPSPATAY DSL 11AGIGGG171 LeuDH(Ti) 69 NAAAGIInGGGVIIGPR-- KDI EARARFIQGN--GRYITAEDVGTVEDlDIIHEET ------- RVYVSGSPAFG PSVPVGYIAAnaSL 171 GluDH (Cs) 114 DSLDFDpn g YRhigpdidVPGDVGAREIGYMYGQYrkvggfyngvLT2arsfggs lvrVMkhendt--l 229 -> <-pd-> <---*7---7...> <> <---- .8- .><1f> <----a9---> <-pg-> <-a7--> <,h-> <--*8---> <pi> <-~9-> <-Pj-> <--*10-> <pk-> * ** * ** * ** LeuDH(X-ray) 172 A----------L-VV-AVSAAVADAGDAVA ---- ADAMPNAIYGVTCDIFAPCAVGAVLNIDFTIPQLA---AAVIAGS 259 LeuDH(Bst) 172 EGWKVVAVGVAYLtJrHISEa-LIVTRIEV ------------------ …---4--AKAVDPNDIYGVEcDIFAPCAIGGIIND IPQL0--AVAGS 259 LeuDH (Ti) 172 EGKVVA9V=LIVVAYELDCIDRLA-LIVQlNKENADRAVQEF__----------------- … …--AEFVHPDKIYDVEIFAPCAmGAIINDTIERLK---CKVVAGS 259 GluDH(Cs) 229 gyiy dpegitteekin ymleragrnkvqdyadkfgvqffpekPWGQKVDID EQAKIvanVKYYIEV 345 <Og> <- -10-> <-ph-> <i> <---11---> <-a12> <j> <pk> <-.13--> <p1> -11-> <p1><-a 12- < … *1><-1--->13… > < - 14---> . ** ** ***** * * * * * LeuiDH(X-ray) 260 AJNL4pRdKYt-VYAPDYVaG AEYGY---NR7AAKVEGDTIEKIFAMHGVP--SYVAADRMAVKARSQFCL

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