M. R. N. Murthy

Structure of beef liver catalase

Abstract The three-dimensional structure of beef liver catalase has been determined to 2.5 a resolution by a combination of isomorphous and molecular replacement techniques. Heavy-atom positions were found using vector search and difference Fourier methods. The tetrameric catalase molecule has 222 symmetry with one of its dyads coincident with a crystallographic 2-fold axis. The known polypeptide sequence has been unambiguously fitted to the electron density map. The heme is well buried in a hydrophobic pocket, 20 A below the surface of the molecule, and accessible through a hydrophobic channel. Residues that line the heme pocket belong to two different subunits. Tyr357 is the proximal heme ligand and the catalytically important residues on the distal side are residues His74 and Asnl47. The tertiary structure consists of four domains: an extended non-globular amino-terminal arm, which stabilizes the quaternary structure; an anti-parallel, eight-stranded β-barrel providing the residues on the distal side of the heme; a rather random “wrapping domain” around the subunit exterior including the proximal heme ligand; and a final λ-helical structure resembling the E, F, G and H helices of the globins.

Triosephosphate isomerase from Plasmodium falciparum: the crystal structure provides insights into antimalarial drug design.

BACKGROUND Malaria caused by the parasite Plasmodium falciparum is a major public health concern. The parasite lacks a functional tricarboxylic acid cycle, making glycolysis its sole energy source. Although parasite enzymes have been considered as potential antimalarial drug targets, little is known about their structural biology. Here we report the crystal structure of triosephosphate isomerase (TIM) from P. falciparum at 2.2 A resolution. RESULTS The crystal structure of P. falciparum TIM (PfTIM), expressed in Escherichia coli, was determined by the molecular replacement method using the structure of trypanosomal TIM as the starting model. Comparison of the PfTIM structure with other TIM structures, particularly human TIM, revealed several differences. In most TIMs the residue at position 183 is a glutamate but in PfTIM it is a leucine. This leucine residue is completely exposed and together with the surrounding positively charged patch, may be responsible for binding TIM to the erythrocyte membrane. Another interesting feature is the occurrence of a cysteine residue at the dimer interface of PfTIM (Cys13), in contrast to human TIM where this residue is a methionine. Finally, residue 96 of human TIM (Ser96), which occurs near the active site, has been replaced by phenylalanine in PfTIM. CONCLUSIONS Although the human and Plasmodium enzymes share 42% amino acid sequence identity, several key differences suggest that PfTIM may turn out to be a potential drug target. We have identified a region which may be responsible for binding PfTIM to cytoskeletal elements or the band 3 protein of erythrocytes; attachment to the erythrocyte membrane may subsequently lead to the extracellular exposure of parts of the protein. This feature may be important in view of a recent report that patients suffering from P. falciparum malaria mount an antibody response to TIM leading to prolonged hemolysis. A second approach to drug design may be provided by the mutation of the largely conserved residue (Ser96) to phenylalanine in PfTIM. This difference may be of importance in designing specific active-site inhibitors against the enzyme. Finally, specific inhibition of PfTIM subunit assembly might be possible by targeting Cys13 at the dimer interface. The crystal structure of PfTIM provides a framework for new therapeutic leads.

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors.

Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that ofMacrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.

The refined structure of beef liver catalase at 2·5 Å resolution

The crystal structure of beef liver catalase [Murthy, Reid, Sicignano, Tanaka & Rossmann (1981). J. Mol. Biol. 152, 465-499] has now been refined by a restrained parameter least-squares method [Konnert & Hendrickson (1980). Acta Cryst. A36, 344-350] with respect to 2.5 A data. Some extra density was discovered during the refinement process. This was interpreted in terms of a bound NADP molecule [Kirkman & Gaetani (1984). Proc. Natl Acad. Sci. USA, 81, 4343-4348; Fita & Rossmann (1985). Proc. Natl Acad. Sci. USA, 82, 1604-1608]. When the noncrystallographic symmetry was imposed as a constraint, the R factor was reduced to 21.2%. However, refinement of the two crystallographic independent subunits gave a final R factor of 19.1%. The refined coordinates have been re-analyzed for main-chain and side-chain hydrogen bonding, charge distribution, secondary structural element interactions, subunit contacts and molecular packing. The fractional accessibility and the temperature-factor variation are also discussed. The oligomerization process is considered in terms of the unusual quaternary structure. The organization of the heme channel and its relation to the enzymes catalytic properties have been discussed elsewhere [Fita & Rossmann (1985). J. Mol. Biol. 185, 21-37].

Structural comparisons of some small spherical plant viruses

The structures of tomato bushy stunt virus, southern bean mosaic virus and satellite tobacco necrosis virus have been compared quantitatively. The organization of the shell domains of tomato bushy stunt virus and southern bean mosaic virus within the icosahedral envelope is identical. The wedge-shaped end of the subunit is closer to the fivefold or quasi-sixfold axes in all three viruses but the packing about the three- and twofold axes is quite different in satellite tobacco necrosis virus as compared to tomato bushy stunt virus or southern bean mosaic virus. The polypeptide folds of these viruses have greatest similarity in the beta-sheet region of the eight-stranded anti-parallel beta-barrel. The largest differences occur in the connecting segments. There is no clear indication of homologous amino acid sequences between southern bean mosaic virus and satellite tobacco necrosis virus. However, there is some conservation of the following functional groups. (1) Threonines and serines at the hexagonal-pentagonal wedge-shaped end of the subunit. (2) Lysines and arginines at the protein-RNA interface. (3) Hydrophobic residues in the cavity within the anti-parallel beta-barrel. (4) An aspartic acid near a site which binds Ca in tomato bushy stunt virus. (5) Ionic interactions in the contacts between fivefold-related subunits. These virus coat protein structures are not as similar to each other as the alpha and beta chains of hemoglobin but have greater likeness to one another than the NAD-binding domains of dehydrogenases or lysozymes from hen egg-white and T4 phage. The surface domains of tomato bushy stunt virus and southern bean mosaic virus are more like each other than like satellite tobacco necrosis virus. A divergent evolutionary tree is proposed on the basis of these observations.

Structure of lobster apo-D-glyceraldehyde-3-phosphate dehydrogenase at 3.0 A resolution.

Abstract Lobster apo-glyceraldehyde-3-phosphate dehydrogenase was prepared by first lowering the pH to 4.8, thus reducing the NAD binding energy, and then separating the enzyme and coenzyme on a Sephadex column. Triclinic crystals were grown from an ammonium sulfate solution at pH 6.2. The apo-structure was initially determined approximately by comparison with the known hologlyceraldehyde-3-phosphate dehydrogenase molecule. The former was then refined using the 222 molecular symmetry with the molecular replacement technique. Only minor conformational differences were observed between apo and holo-glyceraldehyde-3-phosphate dehydrogenase. Trp193 in the “S loop” and the adenine-binding pocket showed the most significant changes.

Evidence for recombination among the tomato leaf curl virus strains/species from Bangalore, India

Summary. Tomato leaf curl virus (ToLCV) belongs to the Begomovirus genus of the family Geminiviridae. These viruses have circular single stranded DNA molecules as their genome encapsidated in icosahedral geminate particles. Generally the Begomoviruses are bipartite with respect to their genomic composition. ToLCV from South India is unique in that only DNA A component has been isolated and sequenced thus far and there is no evidence for the presence of DNA B component. In this communication we report the genomic sequences of DNA A component of two strains of Tomato leaf curl Bangalore virus (ToLCBV), one from Bangalore, ToLCBV [Ban 5] and the other from Kolar (70 kms from Bangalore), ToLCBV [Kolar]. We have examined the possibility of recombination between strains/species that co-exist within the same geographical location. A novel method has been used to analyze the variation of ToLCV sequences reported from Bangalore and to assess the frequency and importance of recombinational events among the strains/species existing in Bangalore. The results indicate that there are potential sites of recombination in AV1, AV2, AC1 and intergenic regions of the viral genome and this accounts for the observed variability in these strains/species.

Structure of sesbania mosaic virus at 3 å resolution

Sesbania mosaic virus (SMV) is an isometric, ss-RNA plant virus found infecting Sesbania grandiflora plants in fields near Tirupathi, South India. The virus particles, which sediment at 116 S at pH 5.5, swell upon treatment with EDTA at pH 7.5 resulting in the reduction of the sedimentation coefficient to 108 S. SMV coat protein amino acid sequence was determined and found to have approximately 60% amino acid sequence identity with that of southern bean mosaic virus (SBMV). The amino terminal 60 residue segment, which contains a number of positively charged residues, is less well conserved between SMV and SBMV when compared to the rest of the sequence. The 3D structure of SMV was determined at 3.0 A resolution by molecular replacement techniques using SBMV structure as the initial phasing model. The icosahedral asymmetric unit was found to contain four calcium ions occurring in inter subunit interfaces and three protein subunits, designated A, B and C. The conformation of the C subunit appears to be different from those of A and B in several segments of the polypeptide. These observations coupled with structural studies on SMV partially depleted of calcium suggest a plausible mechanism for the initiation of the disassembly of the virus capsid.

Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate

In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes.

Crystal Structures of Salmonella typhimurium Biodegradative Threonine Deaminase and Its Complex with CMP Provide Structural Insights into Ligand-induced Oligomerization and Enzyme Activation

Two different pyridoxal 5′-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to α-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB·CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5′-phosphate-dependent enzymes points to structural and mechanistic similarities.