Neutron and light scattering studies of light-harvesting photosynthetic antenna complexes.

Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) have been employed in studying the structural information of various biological systems, particularly in systems without high-resolution structural information available. In this report, we briefly present some principles and biological applications of neutron scattering and DLS, compare the differences in information that can be obtained with small-angle X-ray scattering (SAXS), and then report recent studies of SANS and DLS, together with other biophysical approaches, for light-harvesting antenna complexes and reaction centers of purple and green phototrophic bacteria.

Carbon metabolic pathways in phototrophic bacteria and their broader evolutionary implications.

Photosynthesis is the biological process that converts solar energy to biomass, bio-products, and biofuel. It is the only major natural solar energy storage mechanism on Earth. To satisfy the increased demand for sustainable energy sources and identify the mechanism of photosynthetic carbon assimilation, which is one of the bottlenecks in photosynthesis, it is essential to understand the process of solar energy storage and associated carbon metabolism in photosynthetic organisms. Researchers have employed physiological studies, microbiological chemistry, enzyme assays, genome sequencing, transcriptomics, and (13)C-based metabolomics/fluxomics to investigate central carbon metabolism and enzymes that operate in phototrophs. In this report, we review diverse CO(2) assimilation pathways, acetate assimilation, carbohydrate catabolism, the tricarboxylic acid cycle and some key, and/or unconventional enzymes in central carbon metabolism of phototrophic microorganisms. We also discuss the reducing equivalent flow during photoautotrophic and photoheterotrophic growth, evolutionary links in the central carbon metabolic network, and correlations between photosynthetic and non-photosynthetic organisms. Considering the metabolic versatility in these fascinating and diverse photosynthetic bacteria, many essential questions in their central carbon metabolism still remain to be addressed.

Complete genome sequence of the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus.

BACKGROUND: Chloroflexus aurantiacus is a thermophilic filamentous anoxygenic phototrophic (FAP) bacterium, and can grow phototrophically under anaerobic conditions or chemotrophically under aerobic and dark conditions. According to 16S rRNA analysis, Chloroflexi species are the earliest branching bacteria capable of photosynthesis, and Cfl. aurantiacus has been long regarded as a key organism to resolve the obscurity of the origin and early evolution of photosynthesis. Cfl. aurantiacus contains a chimeric photosystem that comprises some characters of green sulfur bacteria and purple photosynthetic bacteria, and also has some unique electron transport proteins compared to other photosynthetic bacteria. METHODS: The complete genomic sequence of Cfl. aurantiacus has been determined, analyzed and compared to the genomes of other photosynthetic bacteria. RESULTS: Abundant genomic evidence suggests that there have been numerous gene adaptations/replacements in Cfl. aurantiacus to facilitate life under both anaerobic and aerobic conditions, including duplicate genes and gene clusters for the alternative complex III (ACIII), auracyanin and NADH:quinone oxidoreductase; and several aerobic/anaerobic enzyme pairs in central carbon metabolism and tetrapyrroles and nucleic acids biosynthesis. Overall, genomic information is consistent with a high tolerance for oxygen that has been reported in the growth of Cfl. aurantiacus. Genes for the chimeric photosystem, photosynthetic electron transport chain, the 3-hydroxypropionate autotrophic carbon fixation cycle, CO2-anaplerotic pathways, glyoxylate cycle, and sulfur reduction pathway are present. The central carbon metabolism and sulfur assimilation pathways in Cfl. aurantiacus are discussed. Some features of the Cfl. aurantiacus genome are compared with those of the Roseiflexus castenholzii genome. Roseiflexus castenholzii is a recently characterized FAP bacterium and phylogenetically closely related to Cfl. aurantiacus. According to previous reports and the genomic information, perspectives of Cfl. aurantiacus in the evolution of photosynthesis are also discussed. CONCLUSIONS: The genomic analyses presented in this report, along with previous physiological, ecological and biochemical studies, indicate that the anoxygenic phototroph Cfl. aurantiacus has many interesting and certain unique features in its metabolic pathways. The complete genome may also shed light on possible evolutionary connections of photosynthesis.

Temperature and ionic strength effects on the chlorosome light-harvesting antenna complex.

Chlorosomes, the peripheral light-harvesting antenna complex from green photosynthetic bacteria, are the largest and one of the most efficient light-harvesting antenna complexes found in nature. In contrast to other light-harvesting antennas, chlorosomes are constructed from more than 150,000 self-assembled bacteriochlorophylls (BChls) and contain relatively few proteins that play secondary roles. These unique properties have led to chlorosomes as an attractive candidate for developing biohybrid solar cell devices. In this article, we investigate the temperature and ionic strength effects on the viability of chlorosomes from the photosynthetic green bacterium Chloroflexus aurantiacus using small-angle neutron scattering and dynamic light scattering. Our studies indicate that chlorosomes remain intact up to 75 °C and that salt induces the formation of large aggregates of chlorosomes. No internal structural changes are observed for the aggregates. The salt-induced aggregation, which is a reversible process, is more efficient with divalent metal ions than with monovalent metal ions. Moreover, with treatment at 98 °C for 2 min, the bulk of the chlorosome pigments are undamaged, while the baseplate is destroyed. Chlorosomes without the baseplate remain rodlike in shape and are 30-40% smaller than with the baseplate attached. Further, chlorosomes are stable from pH 5.5 to 11.0. Together, this is the first time such a range of characterization tools have been used for chlorosomes, and this has enabled elucidation of properties that are not only important to understanding their functionality but also may be useful in biohybrid devices for effective light harvesting.

SANS investigation of the photosynthetic machinery of Chloroflexus aurantiacus.

Green photosynthetic bacteria harvest light and perform photosynthesis in low-light environments, and contain specialized antenna complexes to adapt to this condition. We performed small-angle neutron scattering (SANS) studies to obtain structural information about the photosynthetic apparatus, including the peripheral light-harvesting chlorosome complex, the integral membrane light-harvesting B808-866 complex, and the reaction center (RC) in the thermophilic green phototrophic bacterium Chloroflexus aurantiacus. Using contrast variation in SANS measurements, we found that the B808-866 complex is wrapped around the RC in Cfx. aurantiacus, and the overall size and conformation of the B808-866 complex of Cfx. aurantiacus is roughly comparable to the LH1 antenna complex of the purple bacteria. A similar size of the isolated B808-866 complex was suggested by dynamic light scattering measurements, and a smaller size of the RC of Cfx. aurantiacus compared to the RC of the purple bacteria was observed. Further, our SANS measurements indicate that the chlorosome is a lipid body with a rod-like shape, and that the self-assembly of bacteriochlorophylls, the major component of the chlorosome, is lipid-like. Finally, two populations of chlorosome particles are suggested in our SANS measurements.

Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum.

The photosynthetic green sulfur bacterium Chlorobaculum tepidum assimilates CO(2) and organic carbon sources (acetate or pyruvate) during mixotrophic growth conditions through a unique carbon and energy metabolism. Using a (13)C-labeling approach, this study examined biosynthetic pathways and flux distributions in the central metabolism of C. tepidum. The isotopomer patterns of proteinogenic amino acids revealed an alternate pathway for isoleucine synthesis (via citramalate synthase, CimA, CT0612). A (13)C-assisted flux analysis indicated that carbons in biomass were mostly derived from CO(2) fixation via three key routes: the reductive tricarboxylic acid (RTCA) cycle, the pyruvate synthesis pathway via pyruvate:ferredoxin oxidoreductase, and the CO(2)-anaplerotic pathway via phosphoenolpyruvate carboxylase. During mixotrophic growth with acetate or pyruvate as carbon sources, acetyl-CoA was mainly produced from acetate (via acetyl-CoA synthetase) or citrate (via ATP citrate lyase). Pyruvate:ferredoxin oxidoreductase converted acetyl-CoA and CO(2) to pyruvate, and this growth-rate control reaction is driven by reduced ferredoxin generated during phototrophic growth. Most reactions in the RTCA cycle were reversible. The relative fluxes through the RTCA cycle were 80∼100 units for mixotrophic cultures grown on acetate and 200∼230 units for cultures grown on pyruvate. Under the same light conditions, the flux results suggested a trade-off between energy-demanding CO(2) fixation and biomass growth rate; C. tepidum fixed more CO(2) and had a higher biomass yield (Y(X/S), mole carbon in biomass/mole substrate) in pyruvate culture (Y(X/S) = 9.2) than in acetate culture (Y(X/S) = 6.4), but the biomass growth rate was slower in pyruvate culture than in acetate culture.

Carbon flow of heliobacteria is related more to clostridia than to the green sulfur bacteria.

The recently discovered heliobacteria are the only Gram-positive photosynthetic bacteria that have been cultured. One of the unique features of heliobacteria is that they have properties of both the photosynthetic green sulfur bacteria (containing the type I reaction center) and Clostridia (forming heat-resistant endospores). Most of the previous studies of heliobacteria, which are strict anaerobes and have the simplest known photosynthetic apparatus, have focused on energy and electron transfer processes. It has been assumed that like green sulfur bacteria, the major carbon flow in heliobacteria is through the (incomplete) reductive (reverse) tricarboxylic acid cycle, whereas the lack of CO(2)-enhanced growth has not been understood. Here, we report studies to fill the knowledge gap of heliobacterial carbon metabolism. We confirm that the CO(2)-anaplerotic pathway is active during phototrophic growth and that isoleucine is mainly synthesized from the citramalate pathway. Furthermore, to our surprise, our results suggest that the oxidative (forward) TCA cycle is operative and more active than the previously reported reductive (reverse) tricarboxylic acid cycle. Both isotopomer analysis and activity assays suggest that citrate is produced by a putative (Re)-citrate synthase and then enters the oxidative (forward) TCA cycle. Moreover, in contrast to (Si)-citrate synthase, (Re)-citrate synthase produces a different isomer of 2-fluorocitrate that is not expected to inhibit the activity of aconitase.

Both forward and reverse TCA cycles operate in green sulfur bacteria.

The anoxygenic green sulfur bacteria (GSBs) assimilate CO(2) autotrophically through the reductive (reverse) tricarboxylic acid (RTCA) cycle. Some organic carbon sources, such as acetate and pyruvate, can be assimilated during the phototrophic growth of the GSBs, in the presence of CO(2) or HCO(3)(-). It has not been established why the inorganic carbonis required for incorporating organic carbon for growth and how the organic carbons are assimilated. In this report, we probed carbon flux during autotrophic and mixotrophic growth of the GSB Chlorobaculum tepidum. Our data indicate the following: (a) the RTCA cycle is active during autotrophic and mixotrophic growth; (b) the flux from pyruvate to acetyl-CoA is very low and acetyl-CoA is synthesized through the RTCA cycle and acetate assimilation; (c) pyruvate is largely assimilated through the RTCA cycle; and (d) acetate can be assimilated via both of the RTCA as well as the oxidative (forward) TCA (OTCA) cycle. The OTCA cycle revealed herein may explain better cell growth during mixotrophic growth with acetate, as energy is generated through the OTCA cycle. Furthermore, the genes specific for the OTCA cycle are either absent or down-regulated during phototrophic growth, implying that the OTCA cycle is not complete, and CO(2) is required for the RTCA cycle to produce metabolites in the TCA cycle. Moreover, CO(2) is essential for assimilating acetate and pyruvate through the CO(2)-anaplerotic pathway and pyruvate synthesis from acetyl-CoA.

Energy metabolism of Heliobacterium modesticaldum during phototrophic and chemotrophic growth.

BACKGROUND: Heliobacterium modesticaldum is a gram-positive nitrogen-fixing phototrophic bacterium that can grow either photoheterotrophically or chemotrophically but not photoautotrophically. Surprisingly, this organism is lacking only one gene for the complete reverse tricarboxylic acid (rTCA) cycle required for autotrophic carbon fixation. Along with the genomic information reported recently, we use multiple experimental approaches in this report to address questions regarding energy metabolic pathways in darkness, CO2 fixation, sugar assimilation and acetate metabolism. RESULTS: We present the first experimental evidence that D-ribose, D-fructose and D-glucose can be photoassimilated by H. modesticaldum as sole carbon sources in newly developed defined growth medium. Also, we confirm two non-autotrophic CO2-fixation pathways utilized by H. modesticaldum: reactions catalyzed by pyruvate:ferredoxin oxidoreductase and phosphoenolpyruvate carboxykinase, and report acetate excretion during phototrophic and chemotrophic growth. Further, genes responsible for pyruvate fermentation, which provides reducing power for nitrogen assimilation, carbon metabolism and hydrogen production, are either active or up-regulated during chemotrophic growth. The discovery of ferredoxin-NADP+ oxidoreductase (FNR) activity in cell extracts provides the reducing power required for carbon and nitrogen metabolisms. Moreover, we show that photosynthetic pigments are produced by H. modesticaldum during the chemotrophic growth, and demonstrate that H. modesticaldum performs nitrogen fixation during both phototrophic and chemotrophic growth. CONCLUSION: Collectively, this report represents the first comprehensive studies for energy metabolism in heliobacteria, which have the simplest known photosynthetic machinery among the entire photosynthetic organisms. Additionally, our studies provide new and essential insights, as well as broaden current knowledge, on the energy metabolism of the thermophilic phototrophic bacterium H. modesticaldum during phototrophic and chemotrophic growth.

Carbohydrate metabolism and carbon fixation in Roseobacter denitrificans OCh114.

The Roseobacter clade of aerobic marine proteobacteria, which compose 10-25% of the total marine bacterial community, has been reported to fix CO(2), although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO(2) assimilation, and amino acid biosynthesis in the phototrophic Roseobacter clade bacterium Roseobacter denitrificans OCh114. We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B(12) (cyanocobalamin). Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination. Through measurements of (13)C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10-15% of protein carbon from CO(2); (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source.

Evidence for conformational movement and radical mechanism in the reaction of 4-thia-L-lysine with lysine 5,6-aminomutase.

We demonstrate that the steady state reaction of lysine 5,6-aminomutase with substrate analogue 4-thia-l-lysine generates a radical intermediate, which accumulates in the enzyme to an electron paramagnetic resonance (EPR) detectable level. EPR line width narrowing of approximately 1 mT due to [4'-(2)H] labeling of the pyridoxal-5'-phosphate (PLP), an isotropic hyperfine coupling of 40 MHz for the proton at C4' of PLP derived from (2)H electron nuclear double resonance (ENDOR) measurement, and spin density delocalization onto the (31)P of PLP realized from observations of the (31)P ENDOR signal provide unequivocal identification of the radical as a substrate-PLP-based species. X- and Q-band EPR spectra fittings demonstrate that this radical is spin coupled with the low spin Co(2+) in cob (II) alamin and the distance between the two species is about 10 A. These results provide direct evidence for the active site motion upon substrate binding, bringing the adenosylcobalamin to the proximity of substrate-PLP for subsequent H-atom abstraction and for the notion that lysine 5,6-aminomutase functions by a radical mechanism. Observation of (2)H-ENDOR signal also provides a reliable hyperfine coupling constant for future comparison with quantum-mechanical-based calculations to gain further insight into the molecular structure of this steady state radical intermediate.

Radical triplets and suicide inhibition in reactions of 4-thia-D- and 4-thia-L-lysine with lysine 5,6-aminomutase.

Lysine 5,6-aminomutase (5,6-LAM) catalyzes the interconversions of D- or L-lysine and the corresponding enantiomers of 2,5-diaminohexanoate, as well as the interconversion of L-beta-lysine and l-3,5-diaminohexanoate. The reactions of 5,6-LAM are 5'-deoxyadenosylcobalamin- and pyridoxal-5'-phosphate (PLP)-dependent. Similar to other 5'-deoxyadenosylcobalamin-dependent enzymes, 5,6-LAM is thought to function by a radical mechanism. No free radicals can be detected by electron paramagnetic resonance (EPR) spectroscopy in reactions of 5,6-LAM with D- or L-lysine or with L-beta-lysine. However, the substrate analogues 4-thia-L-lysine and 4-thia-D-lysine undergo early steps in the mechanism to form two radical species that are readily detected by EPR spectroscopy. Cob(II)alamin and 5'-deoxyadenosine derived from 5'-deoxyadenosylcobalamin are also detected. The radicals are proximal to and spin-coupled with low-spin Co(2+) in cob(II)alamin and appear as radical triplets. The radicals are reversibly formed but do not proceed to stable products, so that 4-thia-D- and L-lysine are suicide inhibitors. Inhibition attains equilibrium between the active Michaelis complex and the inhibited radical triplets. The structure of the transient 4-thia-L-lysine radical is analogous to that of the first substrate-related radical in the putative isomerization mechanism. The second, persistent radical is more stable than the transient species and is assigned as a tautomer, in which a C6(H) of the transient radical is transferred to the carboxaldehyde carbon (C4') of PLP. The persistent radical blocks the active site and inhibits the enzyme, but it decomposes very slowly at

Role of the AcsF protein in Chloroflexus aurantiacus.

The green phototrophic bacteria contain a unique complement of chlorophyll pigments, which self-assemble efficiently into antenna structures known as chlorosomes with little involvement of protein. The few proteins found in chlorosomes have previously been thought to have a primarily structural function. The biosynthetic pathway of the chlorosome pigments, bacteriochlorophylls c, d, and e, is not well understood. In this report, we used spectroscopic, proteomic, and gene expression approaches to investigate the chlorosome proteins of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus. Surprisingly, Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, AcsF, was identified under anaerobic growth conditions. The AcsF protein was found in the isolated chlorosome fractions, and the proteomics analysis suggested that significant portions of the AcsF proteins are not accessible to protease digestion. Additionally, quantitative real-time PCR studies showed that the transcript level of the acsF gene is not lower in anaerobic growth than in semiaerobic growth. Since the proposed enzymatic activity of AcsF requires molecular oxygen, our studies suggest that the roles of AcsF in C. aurantiacus need to be investigated further.

Mismatched dNTP incorporation by DNA polymerase beta does not proceed via globally different conformational pathways.

Understanding how DNA polymerases control fidelity requires elucidation of the mechanisms of matched and mismatched dNTP incorporations. Little is known about the latter because mismatched complexes do not crystallize readily. In this report, we employed small-angle X-ray scattering (SAXS) and structural modeling to probe the conformations of different intermediate states of mammalian DNA polymerase beta (Pol beta) in its wild-type and an error-prone variant, I260Q. Our structural results indicate that the mismatched ternary complex lies in-between the open and the closed forms, but more closely resembles the open form for WT and the closed form for I260Q. On the basis of molecular modeling, this over-stabilization of mismatched ternary complex of I260Q is likely caused by formation of a hydrogen bonding network between the side chains of Gln(260), Tyr(296), Glu(295) and Arg(258), freeing up Asp(192) to coordinate MgdNTP. These results argue against recent reports suggesting that mismatched dNTP incorporations follow a conformational path distinctly different from that of matched dNTP incorporation, or that its conformational closing is a major contributor to fidelity.

Structure and function of 2:1 DNA polymerase.DNA complexes.

DNA polymerases are required for DNA replication and DNA repair in all of the living organisms. Different DNA polymerases are responsible different stages of DNA metabolism, and many of them are multifunctional enzymes. It was generally assumed that the different reactions are catalyzed by the same enzyme molecule. In addition to 1:1 DNA polymerase.DNA complex reported by crystallization studies, 2:1 and higher order DNA polymerase.DNA complexes have been identified in solution studies by various biochemical and biophysical approaches. Further, abundant evidences for the DNA polymerase-DNA interactions in several DNA polymerases suggested that the 2:1 complex represents the more active form. This review describes the current status of this emerging subject and explores their potential in vitro and in vivo functional significance, particularly for the 2:1 complexes of mammalian DNA polymerase beta (Pol beta), the Klenow fragment of E. coli DNA polymerase I (KF), and T4 DNA polymerase.

Solution structures of 2 : 1 and 1 : 1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle X-ray scattering.

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase beta (Pol beta) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol beta-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol beta-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5'-dRP lyase domain of the second Pol beta molecule with the active site of the 1 : 1 Pol beta-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5'-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5'-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol beta-DNA complex enhances the function of Pol beta.

Investigation of the conformational states of Wzz and the Wzz.O-antigen complex under near-physiological conditions.

Chain length determinant protein (Wzz) has been postulated to terminate the polymerization and regulate the chain length of the O-polysaccharide (O-antigen), an important component for constructing lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. The investigation to understand the mechanism of Wzz has been largely slowed down due to lack of structural information. In this report, we have applied small-angle X-ray scattering (SAXS) to study the conformational state and molecular properties of Wzz and the Wzz.O-antigen complex under near-physiological conditions. No concentration-dependent aggregation or structural changes, but repulsive intermolecular interactions between Wzz molecules, are suggested in the concentration series studies. The SAXS studies suggest that Wzz protein appears to be elongated and exists as a tetramer in solution. The reconstructed model built from SAXS data indicates that the middle regime of Wzz, most likely representing the periplasmic domain, contributes to the Wzz oligomerization, which has been proposed to be correlated to the function of Wzz. The immunoblotting analyses also demonstrate that the putative coiled-coil region in the periplasmic region contributes to the oligomerization. Further, the SAXS data corresponding to Wzz and the Wzz.O-antigen complex indicate an apparent substrate (O-antigen)-induced conformational change, consistent with previous circular dichroism studies. Our finding may shed light on the biological mechanism of Wzz as a chain length determinant of O-antigen.

A base triple in the Tetrahymena group I core affects the reaction equilibrium via a threshold effect.

Previous work on group I introns has suggested that a central base triple might be more important for the first rather than the second step of self-splicing, leading to a model in which the base triple undergoes a conformational change during self-splicing. Here, we use the well-characterized L-21 ScaI ribozyme derived from the Tetrahymena group I intron to probe the effects of base-triple disruption on individual reaction steps. Consistent with previous results, reaction of a ternary complex mimicking the first chemical step in self-splicing is slowed by mutations in this base triple, whereas reaction of a ternary complex mimicking the second step of self-splicing is not. Paradoxically, mechanistic dissection of the base-triple disruption mutants indicates that active site binding is weakened uniformly for the 5'-splice site and the 5'-exon analog, mimics for the species bound in the first and second step of self-splicing. Nevertheless, the 5'-exon analog remains bound at the active site, whereas the 5'-splice site analog does not. This differential effect arises despite the uniform destabilization, because the wild-type ribozyme binds the 5'-exon analog more strongly in the active site than in the 5'-splice site analog. Thus, binding into the active site constitutes an additional barrier to reaction of the 5'-splice site analog, but not the 5'-exon analog, resulting in a reduced reaction rate constant for the first step analog, but not the second step analog. This threshold model explains the self-splicing observations without the need to invoke a conformational change involving the base triple, and underscores the importance of quantitative dissection for the interpretation of effects from mutations.

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase.

Lysine 5,6-aminomutase is an adenosylcobalamin and pyridoxal-5'-phosphate-dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of dl-lysine and of l-beta-lysine. We have solved the x-ray structure of a substrate-free form of lysine-5,6-aminomutase from Clostridium sticklandii. In this structure, a Rossmann domain covalently binds pyridoxal-5'-phosphate by means of lysine 144 and positions it into the putative active site of a neighboring triosephosphate isomerase barrel domain, while simultaneously positioning the other cofactor, adenosylcobalamin, approximately 25 A from the active site. In this mode of pyridoxal-5'-phosphate binding, the cofactor acts as an anchor, tethering the separate polypeptide chain of the Rossmann domain to the triosephosphate isomerase barrel domain. Upon substrate binding and transaldimination of the lysine-144 linkage, the Rossmann domain would be free to rotate and bring adenosylcobalamin, pyridoxal-5'-phosphate, and substrate into proximity. Thus, the structure embodies a locking mechanism to keep the adenosylcobalamin out of the active site and prevent radical generation in the absence of substrate.