Improving the catalytic activity of a thermophilic enzyme at low temperatures

Enzymes from thermophilic organisms often are barely active at low temperatures. To obtain a better understanding of this sluggishness, we used DNA shuffling to mutagenize the trpC gene, which encodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutants producing more active protein variants were selected by genetic complementation of an Escherichia coli mutant bearing a trpC deletion. Single amino acid changes and combinations of these changes improved growth appreciably. Five singly and doubly altered protein variants with changes at the N- and C-termini, or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymatic catalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purified variant proteins correlated with the corresponding growth rates, showing that the turnover number was the selected trait. Although the affinities for both the substrate and the product decreased appreciably in most protein variants, these defects were offset by the accumulation of high levels of the enzyme's substrate. Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme's turnover number at low temperatures is limited by the dissociation of the enzyme-product complex. In contrast, representative protein variants bind and release the product far more rapidly, shifting the bottleneck to the preceding chemical step. The turnover number of the parental enzyme increases with temperature, suggesting that its structural rigidity is responsible for its poor catalytic activity at low temperatures. In support of this interpretation, the rate of trypsinolysis or of thermal denaturation is accelerated significantly in the activated protein variants.     

The 24-kDa iron-sulphur subunit of complex I is required for enzyme activity.

We have cloned the nuclear gene encoding the 24-kDa iron-sulphur subunit of complex I from Neurospora crassa. The gene was inactivated in vivo by repeat-induced point-mutations, and mutant strains lacking the 24-kDa protein were isolated. Mutant nuo24 appears to assemble an almost intact complex I only lacking the 24-kDa subunit. However, we also found reduced levels of the NADH-binding, 51-kDa subunit of the enzyme. Surprisingly, the complex I from the nuo24 strain lacks NADH:ferricyanide reductase activity. In agreement with this, the respiration of intact mitochondria or mitochondrial membranes from the mutant strain is insensitive to rotenone inhibition. These results suggest that the nuo24 complex is not functioning in electron transfer and the 24-kDa protein is absolutely required for complex I activity. This phenotype may explain the findings that the 24-kDa iron-sulphur protein is reduced or absent in human mitochondrial diseases. In addition, selected substitutions of cysteine to alanine residues in the 24-kDa protein suggest that binding of the iron-sulphur centre is a requisite for protein assembly.     

The RNA-binding PUA domain of archaeal tRNA-guanine transglycosylase is not required for archaeosine formation

Bacterial tRNA-guanine transglycosylase (TGT) replaces the G in position 34 of tRNA with preQ(1), the precursor to the modified nucleoside queuosine. Archaeal TGT, in contrast, substitutes preQ(0) for the G in position 15 of tRNA as the first step in archaeosine formation. The archaeal enzyme is about 60% larger than the bacterial protein; a carboxyl-terminal extension of 230 amino acids contains the PUA domain known to contact the four 3'-terminal nucleotides of tRNA. Here we show that the C-terminal extension of the enzyme is not required for the selection of G15 as the site of base exchange; truncated forms of Pyrococcus furiosus TGT retain their specificity for guanine exchange at position 15. Deletion of the PUA domain causes a 4-fold drop in the observed k(cat) (2.8 x 10(-3) s(-1)) and results in a 75-fold increased K(m) for tRNA(Asp)(1.2 x 10(-5) m) compared with full-length TGT. Mutations in tRNA(Asp) altering or abolishing interactions with the PUA domain can compete with wild-type tRNA(Asp) for binding to full-length and truncated TGT enzymes. Whereas the C-terminal domains do not appear to play a role in selection of the modification site, their relevance for enzyme function and their role in vivo remains to be discovered.     

The zinc finger domain of the archaeal minichromosome maintenance protein is required for helicase activity

The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum Delta H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX(2)CX(n)CX(2)C (C(4)) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif.     

The catalytic core of an archaeal 2-oxoacid dehydrogenase multienzyme complex is a 42-mer protein assembly

The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes shares a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Thermoplasma acidophilum, a thermophilic archaeon, the homo-trimers assemble into a unique 42-mer oblate spheroid. Analytical equilibrium centrifugation and small-angle X-ray scattering analyses confirm that this catalytically active 1.08 MDa assembly exists as a single species in solution, forming a hollow spheroid with a maximum diameter of 220 ?. In this paper we show that a monodisperse macromolecular assembly, built from identical subunits in non-identical environments, forms an irregular protein shell via non-equivalent interactions. This unusually irregular protein shell, combining cubic and dodecahedral geometrical elements, expands on the concept of quasi-equivalence as a basis for understanding macromolecular assemblies by showing that cubic point group symmetry is not a physical requirement in multienzyme assembly. These results extend our basic knowledge of protein assembly and greatly expand the number of possibilities to manipulate self-assembling biological complexes to be utilized in innovative nanotechnology applications.     

The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1-E2 complex formation

Apg7p/Cvt2p, a protein-activating enzyme, is essential for both the Apg12p-Apg5p conjugation system and the Apg8p membrane targeting in autophagy and cytoplasm-to-vacuole targeting in the yeast Saccharomyces cerevisiae. Similar to the ubiquitin-conjugating system, both Apg12p and Apg8p are activated by Apg7p, an E1-like enzyme. Apg12p is then transferred to Apg10p, an E2-like enzyme, and conjugated with Apg5p, whereas Apg8p is transferred to Apg3p, another E2-like enzyme, followed by conjugation with phosphatidylethanolamine. Evidence is presented here that Apg7p forms a homodimer with two active-site cysteine residues via the C-terminal region. The dimerization of Apg7p is independent of the other Apg proteins and facilitated by overexpressed Apg12p. The C-terminal 123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are sufficient for its dimerization, where there is neither an ATP binding domain nor an active-site cysteine essential for its E1 activity. The deletion of its carboxyl 40 amino acids (residues 591-630 out of 630 amino acids) results in several defects of not only Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an active-site cysteine. Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the interaction of Apg7p with Apg3p to form the E1-E2 complex for Apg8p. These results suggest that Apg7p forms a homodimer via the C-terminal region and that the C-terminal region is essential for both the activity of the E1 enzyme for Apg12p and Apg8p as well as the formation of an E1-E2 complex for Apg8p.     

The Fanconi anemia core complex is required for efficient point mutagenesis and Rev1 foci assembly

Fanconi anemia (FA) is a chromosome instability syndrome characterized by congenital abnormalities, cellular hypersensitivity to DNA crosslinking agents, and heightened cancer risk. Eight of the thirteen identified FA genes encode subunits of a nuclear FA core complex that monoubiquitinates FANCD2 and FANCI to maintain genomic stability in response to replication stress. The FA pathway has been implicated in the regulation of error-prone DNA damage tolerance via an undefined molecular mechanism. Here, we show that the FA core complex is required for efficient spontaneous and UVC-induced point mutagenesis, independently of FANCD2 and FANCI. Consistent with the observed hypomutability of cells deficient in the FA core complex, we also demonstrate that these cells are impaired in the assembly of the error-prone translesion DNA synthesis polymerase Rev1 into nuclear foci. Consistent with a role downstream of the FA core complex and like known FA proteins, Rev1 is required to prevent DNA crosslinker-induced chromosomal aberrations in human cells. Interestingly, proliferating cell nuclear antigen (PCNA) monoubiquitination, known to contribute to Rev1 recruitment, does not require FA core complex function. Our results suggest a role for the FA core complex in regulating Rev1-dependent DNA damage tolerance independently of FANCD2, FANCI, and PCNA monoubiquitination.     

The C-terminal alpha helix of Tn5 transposase is required for synaptic complex formation

An important step in Tn5 transposition requires transposase-transposase homodimerization to form a synaptic complex competent for cleavage of transposon DNA free from the flanking sequence. We demonstrate that the C-terminal helix of Tn5 transposase (residues 458-468 of 476 total amino acids) is required for synaptic complex formation during Tn5 transposition. Specifically, deletion of eight amino acids or more from the C terminus greatly reduces or abolishes synaptic complex formation in vitro. Due to this impaired synaptic complex formation, transposases lacking eight amino acids are also defective in the cleavage step of transposition. Interactions within the synaptic complex dimer interface were investigated by site-directed mutagenesis, and residues required for synaptic complex formation include amino acids comprising the dimer interface in the Tn5 inhibitor x-ray crystal structure dimer. Because the crystal structure dimer was hypothesized to be the inhibitory complex and not a synaptic complex, this result was surprising. Based on these data, models for both in vivo and in vitro synaptic complex formation are presented.     

The insE open reading frame of IS1 is not required for formation of cointegrates.

The role of the insE open reading frame in transposition of IS1 was reexamined by using an insE nonsense mutation that does not alter the amino acid sequence of InsA inhibitor or InsAB transposase. The mutant was active in all strains tested, showing that insE is not essential for formation of cointegrates.     

Nek2A kinase stimulates centrosome disjunction and is required for formation of bipolar mitotic spindles

Nek2A is a cell cycle-regulated kinase of the never in mitosis A (NIMA) family that is highly enriched at the centrosome. One model for Nek2A function proposes that it regulates cohesion between the mother and daughter centriole through phosphorylation of C-Nap1, a large coiled-coil protein that localizes to centriolar ends. Phosphorylation of C-Nap1 at the G2/M transition may trigger its displacement from centrioles, promoting their separation and subsequent bipolar spindle formation. To test this model, we generated tetracycline-inducible cell lines overexpressing wild-type and kinase-dead versions of Nek2A. Live cell imaging revealed that active Nek2A stimulates the sustained splitting of interphase centrioles indicative of loss of cohesion. However, this splitting is accompanied by only a partial reduction in centriolar C-Nap1. Strikingly, induction of kinase-dead Nek2A led to formation of monopolar spindles with unseparated spindle poles that lack C-Nap1. Furthermore, kinase-dead Nek2A interfered with chromosome segregation and cytokinesis and led to an overall change in the DNA content of the cell population. These results provide the first direct evidence in human cells that Nek2A function is required for the correct execution of mitosis, most likely through promotion of centrosome disjunction. However, they suggest that loss of centriole cohesion and C-Nap1 displacement may be distinct mitotic events.     

The Schizosaccharomyces pombe Cdc7 protein kinase required for septum formation is a client protein of Cdc37

Cdc37 is an essential molecular chaperone found in fungi and metazoa whose main specificity is for certain protein kinases. Cdc37 can act as an Hsp90 cochaperone or alone; in yeasts, the interaction with Hsp90 is weak and appears not to be essential for Cdc37 function. Numerous genetic interactions between Cdc37 and likely client proteins have been observed in yeasts, but biochemical confirmation has been reported in only a few cases. We and others have generated and characterized temperature-sensitive cdc37 alleles in S. pombe and have used them to investigate the cellular roles of Cdc37: previous work has shown that mitotic Cdc2 is a major client. In this paper, we describe a screen for mutations synthetically lethal with a cdc37ts mutant with the aim of identifying genes encoding further client proteins of Cdc37. Ten such strains were isolated, and genomic libraries were screened for rescuing plasmids. In one case, a truncated cdc7 gene was identified. Further experiments showed that the mutation in this strain was indeed in cdc7. Cdc7 is a protein kinase required for septum initiation, and we show that its kinase activity is greatly reduced when Cdc37 function is impaired. Cdc7 normally locates to the spindle pole body during mitosis, and this appears to be unaffected in the cdc37ts mutant. Other evidence suggests that, in addition to mitosis and septum initiation, Cdc37 may also be required for septum cleavage.     

ATP is required for correct folding and disulfide bond formation of rotavirus VP7

Rotavirus is one of very few viruses that utilize the endoplasmic reticulum (ER) for assembly, and therefore it has been used as an attractive model to study ER-associated protein folding. In this study, we have examined the requirements for metabolic energy (ATP) for correct folding of the luminal and ER-associated VP7 of rotavirus. We found that VP7 rapidly misfolds in an energy-depleted milieu and is not degraded within 60 min. We also found that VP7 attained a stable minimum-energy state soon after translation in the ER. Most surprisingly, energy-misfolded VP7 could be recovered and establish correct disulfide bonds and antigenicity following a shift to an ATP-rich milieu. Using a Semliki Forest virus expression system, we observed that VP7 requires ATP and cellular, but not viral, factors for correct disulfide bond formation. Our results show for the first time that the disulfide bond formation of rotavirus VP7 is an ATP-dependent process. It has previously been shown that chaperones hydrolyze ATP during interaction with newly synthesized polypeptides and prevent nonproductive intra- and intermolecular interactions. The most reasonable explanation for the energy requirement of VP7 is thus a close interaction during folding with an ATP-dependent chaperone, such as BiP (Grp78), and possibly with protein disulfide isomerase. Taken together, our observations provide new information about folding of ER-associated proteins in general and rotavirus VP7 in particular.     

MeaB is a component of the methylmalonyl-CoA mutase complex required for protection of the enzyme from inactivation

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA. In humans, deficiencies in the mutase lead to methylmalonic aciduria, a rare disease that is fatal in the first year of life. Such inherited deficiencies can result from mutations in the mutase structural gene or from mutations that impair the acquisition of cobalamins. Recently, a human gene of unknown function, MMAA, has been implicated in methylmalonic aciduria (Dobson, C. M., Wai, T., Leclerc, D., Wilson, A., Wu, X., Dore, C., Hudson, T., Rosenblatt, D. S., and Gravel, R. A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15554-15559). MMAA orthologs are widespread in bacteria, archaea, and eukaryotes. In Methylobacterium extorquens AM1, a mutant defective in the MMAA homolog meaB was unable to grow on C(1) and C(2) compounds because of the inability to convert methylmalonyl-CoA to succinyl-CoA (Korotkova N., Chistoserdova, L., Kuksa, V., and Lidstrom, M. E. (2002) J. Bacteriol. 184, 1750-1758). Here we demonstrate that this defect is not due to the absence of adenosylcobalamin but due to an inactive form of methylmalonyl-CoA mutase. The presence of active mutase in double mutants defective in MeaB and in the synthesis of either R-methylmalonyl-CoA or adenosylcobalamin indicates that MeaB is necessary for protection of mutase from inactivation during catalysis. MeaB and methylmalonyl-CoA mutase from M. extorquens were cloned and purified in their active forms. We demonstrated that MeaB forms a complex with methylmalonyl-CoA mutase and stimulates in vitro mutase activity. These results support the hypothesis that MeaB functions to protect methylmalonyl-CoA mutase from irreversible inactivation.     

Ability of tetrahydrobiopterin analogues to support catalysis by inducible nitric oxide synthase: formation of a pterin radical is required for enzyme activity

Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H(4)B) or tetrahydrobiopterin analogues (5-methyl-H(4)B and 4-amino-H(4)B), and the ability of bound 5-methyl-H(4)B and 4-amino-H(4)B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme domain (HDiNOS) was examined. In a single turnover with HDiNOS, 5-methyl-H(4)B forms a very stable radical, 5-methyl-H(3)B(*), that accumulates in the arginine reaction to approximately 60% of the HDiNOS concentration and decays approximately 400-fold more slowly than H(3)B(*) (0.0003 vs 0.12 s(-1)). The amount of radical (5-methyl-H(3)B(*) or H(3)B(*)) observed in the NHA reaction is very small (<3% of HDiNOS). The activity of 5-methyl-H(4)B-saturated FLiNOS and HDiNOS is similar to that when H(4)B is bound: arginine is hydroxylated to NHA, and NHA is oxidized exclusively to citrulline and (*)NO. A pterin radical was not observed with 4-amino-H(4)B- or pterin-free HDiNOS with either substrate. The catalytic activity of 4-amino-H(4)B-bound FLiNOS and HDiNOS resembles that of pterin-free iNOS: the hydroxylation of arginine is very unfavorable (<2% that of H(4)B-bound iNOS), and NHA is oxidized to a mixture of amino acid products (citrulline and cyanoornithine) and NO(-) rather than (*)NO. These results demonstrate that the bound pterin cofactor undergoes a one-electron oxidation (to form a pterin radical), which is essential to its ability to support normal NOS turnover. Although binding of H(4)B also stabilizes the NOS structure and active site, the most critical role of the pterin cofactor in NOS appears to be in electron transfer.     

Structural specificity in a FGF7-affinity purified heparin octasaccharide required for formation of a complex with FGF7 and FGFR2IIIb

Variations in sulfation of heparan sulfate (HS) affect interaction with FGF, FGFR, and FGF-HS-FGFR signaling complexes. Whether structurally distinct HS motifs are at play is unclear. Here we used stabilized recombinant FGF7 as a bioaffinity matrix to purify size-defined heparin oligosaccharides. We show that only 0.2%-4% of 6 to 14 unit oligosaccharides, respectively, have high affinity for FGF7 based on resistance to salt above 0.6M NaCl. The high affinity fractions exhibit highest specific activity for interaction with FGFR2IIIb and formation of complexes of FGF7-HS-FGFR2IIIb. The majority fractions with moderate (0.30-0.6M NaCl), low (0.14-0.30M NaCl) or no affinity at 0.14M NaCl for FGF7 supported no complex formation. The high affinity octasaccharide mixture exhibited predominantly 7- and 8-sulfated components (7,8-S-OctaF7) and formed FGF7-HS-FGFR2IIIb complexes with highest specific activity. Deduced disaccharide analysis indicated that 7,8-S-OctaF7 comprised of DeltaHexA2SGlcN6S in a 2:1 ratio to a trisulfated and a variable unsulfated or monosulfated disaccharide. The inactive octasaccharides with moderate affinity for FGF7 were much more heterogenous and highly sulfated with major components containing 11 or 12 sulfates comprised of predominantly trisulfated disaccharides. This suggests that a rare undersulfated motif in which sulfate groups are specifically distributed has highest affinity for FGF7. The same motif also exhibits structural requirements for high affinity binding to dimers of FGFR2IIIb prior to binding FGF7 to form FGF7-HS-FGFR2IIIb complexes. In contrast, the majority of more highly sulfated HS motifs likely play FGFR-independent roles in stability and control of access of FGF7 to FGFR2IIIb in the tissue matrix.