William Melik-Adamyan

Three-dimensional structure of catalase from Penicillium vitale at 2.0 Å resolution

The three-dimensional structure analysis of crystalline fungal catalase from Penicillium vitale has been extended to 2.0 A resolution. The crystals belong to space group P3(1)21, with the unit cell parameters of a = b = 144.4 A and c = 133.8 A. The asymmetric unit contains half a tetrameric molecule of 222 symmetry. Each subunit is a single polypeptide chain of approximately 670 amino acid residues and binds one heme group. The amino acid sequence has been tentatively determined by computer graphics model building (using the FRODO system) and comparison with the known sequence of beef liver catalase. The atomic model has been refined by the Hendrickson & Konnert (1981) restrained least-squares program against 68,000 reflections between 5 A and 2 A resolution. The final R-factor is 0.31 after 24 refinement cycles. The secondary and tertiary structure of the catalase has been analyzed.

Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 Å resolution

The three‐dimensional crystal structure of catalase from Micrococcus lysodeikticus has been solved by multiple isomorphous replacement and refined at 1.5 Å resolution. The subunit of the tetrameric molecule of 222 symmetry consists of a single polypeptide chain of about 500 amino acid residues and one haem group. The crystals belong to space group P42212 with unit cell parameters a = b = 106,7 Å, c = 106,3 Å, and there is one subunit of the tetramer. per asymmetric unit. The amino acid sequence has been tentatively determined by computer graphics model building and comparison with the known three‐dimensional structure of beef liver catalase and sequences of several other catalases. The atomic model has been refined by Hendrickson and Konnerts least‐squares minimisation against 94,315 reflections between 8 Å and 1.5 Å. The final model consists or 3,977 non‐hydrogen atoms of the protein and haem group, 426 water molecules and ones sulphate ion. The secondary and tertiary sructures of the bacterial catalase have been analyzed and a comparison with the structure of beef liver catalase has been made.

Structure of the Heme d of Penicillium vitale and Escherichia coli Catalases

A heme d prosthetic group with the configuration of a cis-hydroxychlorin -spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the α--meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.

The structures of Micrococcus lysodeikticus catalase, its ferryl intermediate (compound II) and NADPH complex

The crystal structure of the bacterial catalase from Micrococcus lysodeikticus has been refined using the gene-derived sequence both at 0.88 A resolution using data recorded at 110 K and at 1.5 A resolution with room-temperature data. The atomic resolution structure has been refined with individual anisotropic atomic thermal parameters. This has revealed the geometry of the haem and surrounding protein, including many of the H atoms, with unprecedented accuracy and has characterized functionally important hydrogen-bond interactions in the active site. The positions of the H atoms are consistent with the enzymatic mechanism previously suggested for beef liver catalase. The structure reveals that a 25 A long channel leading to the haem is filled by partially occupied water molecules, suggesting an inherent facile access to the active site. In addition, the structures of the ferryl intermediate of the catalase, the so-called compound II, at 1.96 A resolution and the catalase complex with NADPH at 1.83 A resolution have been determined. Comparison of compound II and the resting state of the enzyme shows that the binding of the O atom to the iron (bond length 1.87 A) is associated with increased haem bending and is accompanied by a distal movement of the iron and the side chain of the proximal tyrosine. Finally, the structure of the NADPH complex shows that the cofactor is bound to the molecule in an equivalent position to that found in beef liver catalase, but that only the adenine part of NADPH is visible in the present structure.

The structure of SAICAR synthase: an enzyme in the de novo pathway of purine nucleotide biosynthesis.

BACKGROUND The biosynthesis of key metabolic components is of major interest to biologists. Studies of de novo purine synthesis are aimed at obtaining a deeper understanding of this central pathway and the development of effective chemotherapeutic agents. Phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase catalyses the seventh step out of ten in the biosynthesis of purine nucleotides. To date, only one structure of an enzyme involved in purine biosynthesis has been reported: adenylosuccinate synthetase, which catalyses the first committed step in the synthesis of AMP from IMP. RESULTS We report the first three-dimensional structure of a SAICAR synthase, from Saccharomyces cerevisiae. It is a monomer with three domains. The first two domains consist of antiparallel beta sheets and the third is composed of two alpha helices. There is a long deep cleft made up of residues from all three domains. Comparison of SAICAR synthases by alignment of their sequences reveals a number of conserved residues, mostly located in the cleft. The presence of two sulphate ions bound in the cleft, the structure of SAICAR synthase in complex with ATP and a comparison of this structure with that of other ATP-dependent proteins point to the interdomain cleft as the location of the active site. CONCLUSIONS The topology of the first domain of SAICAR synthase resembles that of the N-terminal domain of proteins belonging to the cyclic AMP-dependent protein kinase family. The fold of the second domain is similar to that of members of the D-alanine:D-alanine ligase family. Together these enzymes form a new superfamily of mononucleotide-binding domains. There appears to be no other enzyme, however, which is composed of the same combination of three domains, with the individual topologies found in SAICAR synthase.

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at ∼2.0‐Å resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H2O2 passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3‐Å resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222‐point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His–Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled. Proteins 2001;44:270–281.

Structure of the Clade 1 catalase, CatF of Pseudomonas syringae, at 1.8 Å resolution

Catalase CatF of Pseudomonas syringae has been identified phylogenetically as a clade 1 catalase, closely related to plant catalases, a group from which no structure has been determined. The structure of CatF has been refined at 1.8 Å resolution by using X‐ray synchrotron data collected from a crystal flash‐cooled with liquid nitrogen. The crystallographic agreement factors R and Rfree are, respectively, 18.3% and 24.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222‐point group symmetry. The crystallized enzyme is a homotetramer of subunits with 484 residues, some 26 residues shorter than predicted from the DNA sequence. Mass spectrometry analysis confirmed the absence of 26 N‐terminal residues, possibly removed by a periplasmic transport system. The core structure of the CatF subunit was closely related to seven other catalases with root‐mean‐square deviations (RMSDs) of 368 core Cα atoms of 0.99–1.30 Å. The heme component of CatF is heme b in the same orientation that is found in Escherichia coli hydroperoxidase II, an orientation that is flipped 180° with respect the orientation of the heme in bovine liver catalase. NADPH is not found in the structure of CatF because key residues required for nucleotide binding are missing; 2129 water molecules were refined into the model. Water occupancy in the main or perpendicular channel of CatF varied among the four subunits from two to five in the region between the heme and the conserved Asp150. A comparison of the water occupancy in this region with the same region in other catalases reveals significant differences among the catalases. Proteins 2003;50:423–436.

Structure of Catalases

Catalase (hydrogen peroxide: hydrogen peroxide oxidoreductase, EC 1.11.1.6) is found in virtually all aerobic organisms. It employs a two-electron-transfer mechanism to disproportionate hydrogen peroxide into molecular oxygen and water (Deisseroth and Dounce 1970). These enzymes serve, in part, to protect the cell from the toxic effects of small peroxides. However, the entire range of biological functions of catalases remains unclear. Two types of catalases are known: Mn-catalases and heme-containing catalases. Mn-catalases, only present in certain prokaryotes, are non-heme hexameric enzymes with an “all-α” fold (Barynin et al. 1986). In contrast, heme-containing catalases, which are widespread, are homotetramers with molecular weights ranging from about 200,000 to 350,000. The three-dimensional crystal structures of five of these heme-containing catalases (Table 1) have been reported at almost atomic resolution. These include members from two eukaryote and from three prokaryote catalases: (1) from the fungus Penicillium vitale (PVC) (Vainshtein et al. 1981Vainshtein et al. 1986; G. Murshudov et al., unpubl.); (2) the mammalian catalase from beef liver (BLC) (Murthy et al. 1981; Fita et al. 1986); (3) from Micrococcus lysodeikticus (MLC) (Murshudov et al. 1992 and unpubl.), (4) from a peroxide-resistant mutant of Proteus mirabilis (PMC_PR) (Gouet et al. 1995); and (5) HPII catalase from E. coli (Bravo et al. 1995). The α+β fold of the subunits and the molecular organization in all these heme catalase structures are unique among proteins and, in particular, present striking differences from heme peroxidases. In catalases the four heme groups are deeply buried inside the molecule due to complex intersubunit interactions...

Crystal Structure of the Protealysin Precursor INSIGHTS INTO PROPEPTIDE FUNCTION

Protealysin (PLN) belongs to the M4 family of peptidases that are commonly known as thermolysin-like proteases (TLPs). All TLPs are synthesized as precursors containing N-terminal propeptides. According to the primary structure of the N-terminal propeptides, the family is divided into two distinct groups. Representatives of the first group including thermolysin and all TLPs with known three-dimensional structures have long prosequences (∼200 amino acids). Enzymes of the second group, whose prototype is protealysin, have short (∼50 amino acids) propeptides. Here, we present the 1.8 Å crystal structure of PLN precursor (proPLN), which is the first three-dimensional structure of a TLP precursor. Whereas the structure of the catalytic domain of proPLN is similar overall to previously reported structures of mature TLPs, it has specific features, including the absence of calcium-binding sites, and different structures of the N-terminal region and substrate-binding site. PLN propeptide forms a separate domain in the precursor and likely acts as an inhibitor that blocks the substrate-binding site and fixes the “open” conformation of the active site, which is unfavorable for catalysis. Furthermore the conserved PPL motif identified in our previous studies directly interacts with the S′ subsites of the active center being a critical element of the propeptide-catalytic domain interface. Comparison of the primary structures of TLPs with short propeptides suggests that the specific features revealed in the proPLN crystal structure are typical for all protealysin-like enzymes. Thus, such proteins can be considered as a separate subfamily of TLPs.

Structure of the C-terminal domain of the catalase-peroxidase KatG from Escherichia coli.

Catalase-peroxidases or KatGs, the apparent in vivo activators of the anti-tubercular pro-drug isoniazid, are active as homodimers, each subunit having two distinct but sequence- and structure-related domains. The N-terminal domain contains the haem group and is catalytically active, while the C-terminal domain lacks the cofactor. The C-terminal domain of KatG from Escherichia coli is expressed as a soluble protein which has been crystallized in triclinic, orthorhombic and tetragonal crystal forms. Packing in the orthorhombic crystals, with eight molecules in the asymmetric unit, follows the pattern of commensurate modulated structures, which explains the diversity of pseudo-origin peaks observed in the native Patterson map. The different crystal forms arise from variations in the length and sequence of the N-terminal extensions in the different constructs. Despite the variability in the N-terminal region, the overall domain conformations beginning with Pro437 are very similar both to each other and to the C-terminal domains within the native structures of the KatGs from Haloarcula marismortui and Burkholderia pseudomallei. Some structural reorganization in the C-terminal domain relative to the N-terminal domain has evolved to compensate for the absence of the haem group. A high percentage of the residues in the C-terminal domains of KatG proteins from different sources are highly conserved and these residues are spread uniformly throughout the domain. The easily folded nature and retention of structure in the C-terminal domain suggests that it may serve as a platform for the folding of the N-terminal domain and for stabilization of the molecular dimer.