Alessio Peracchi

A genomic overview of pyridoxal‐phosphate‐dependent enzymes

Enzymes that use the cofactor pyridoxal phosphate (PLP) constitute a ubiquitous class of biocatalysts. Here, we analyse their variety and genomic distribution as an example of the current opportunities and challenges for the study of protein families. In many free‐living prokaryotes, almost 1.5% of all genes code for PLP‐dependent enzymes, but in higher eukaryotes the percentage is substantially lower, consistent with these catalysts being involved mainly in basic metabolism. Assigning the function of PLP‐dependent enzymes simply on the basis of sequence criteria is not straightforward because, as a consequence of their common mechanistic features, these enzymes have intricate evolutionary relationships. Thus, many genes for PLP‐dependent enzymes remain functionally unclassified, and several of them might encode undescribed catalytic activities. In addition, PLP‐dependent enzymes often show catalytic promiscuity (that is, a single enzyme catalyses different reactions), implying that an organism can have more PLP‐dependent activities than it has genes for PLP‐dependent enzymes. This observation presumably applies to many other classes of protein‐encoding genes.

Involvement of a specific metal ion in the transition of the hammerhead ribozyme to its catalytic conformation

Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by thepro-R p oxygen of phosphate 9 and by N7 of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties of a hammerhead containing a unique phosphorothioate at position 9. The R p-, but notS p-, phosphorothioate reduces the cleavage rate by 103-fold, and the rate can be fully restored by addition of low concentrations of Cd2+, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location ∼20 Å from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd2+ binds with a K d of 25 μm in the ground state and a K d of 2.5 nm in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.

DNA catalysis: potential, limitations, open questions.

The “RNA world” hypothesis, initially formulated in the 1960s, states that life evolved from some protobiotic system in which RNA molecules were capable of self-replication and of a rudimentary form of metabolism. The hypothesis is consistent with circumstantial evidence, and it was strongly supported by the discovery, in the early 1980s, of catalytic RNA molecules (ribozymes). In fact, it is now known that catalytic RNAs play key roles even in extant organisms, where several crucial processes—including RNA splicing and protein synthesis—are carried out by ribozymes. RNA appears to be a plausible candidate as the progenitor biopolymer, since it can both carry genetic information and assume a great variety of tertiary structures and, hence, of functions. However, other RNA-like polymers could, in principle, play the same dual role, most notably DNA, which also is a fundamental component of modern living organisms. Is it then possible to hypothesize that a “DNA world” existed or that it could exist? Or, if RNA (and not DNA) is really at the origin of life, does this mirror simply an accident of evolution or does it hint at some more profound, inherent differences between the two polynucleotides? These kinds of questions have begun to be addressed experimentally in recent years, during which time a large amount of research has shown that single-stranded DNA, much like single-stranded RNA, can fold into structures capable of molecular recognition and catalysis. This Minireview summarizes our current understanding of the catalytic capabilities of DNA, highlighting the theoretical and practical implications of this topic and stressing some of the questions that remain open in the field.

Exploring and exploiting allostery: Models, evolution, and drug targeting

The concept of allostery was elaborated almost 50years ago by Monod and coworkers to provide a framework for interpreting experimental studies on the regulation of protein function. In essence, binding of a ligand at an allosteric site affects the function at a distant site exploiting protein flexibility and reshaping protein energy landscape. Both monomeric and oligomeric proteins can be allosteric. In the past decades, the behavior of allosteric systems has been analyzed in many investigations while general theoretical models and variations thereof have been steadily proposed to interpret the experimental data. Allostery has been established as a fundamental mechanism of regulation in all organisms, governing a variety of processes that range from metabolic control to receptor function and from ligand transport to cell motility. A number of studies have shed light on how evolutionary pressures have favored and molded the development of allosteric features in specific macromolecular systems. The widespread occurrence of allostery has been recently exploited for the development and design of allosteric drugs that bind to either physiological or non-physiological allosteric sites leading to gain of function or loss of function. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Time course of chemical and structural events in protein crystals measured by microspectrophotometry

The functional properties of proteins in the crystalline state have been investigated over the past 30 years by a variety of methods, including single crystal polarized absorption spectroscopy. This technique has provided information on the accumulation and equilibrium distribution of protein-ligand complexes in the crystal and, in a few cases, on the rates of interconversion of catalytic intermediates. It has been possible to detect synergistic effects in the binding of different ligands, cooperativity and half-site reactivity and even formation of active multiprotein complexes, obtained by diffusion of one small protein in the pre-formed crystals of the other. Lattice interactions restrain the conformational transitions of some proteins existing in multiple states in solution. The crystal offers the unique opportunity to analyse not only the structure but also the function of a single form of the protein. The relevance of these data to the planning and interpretation of structural studies, especially in the perspectives of time-resolved crystallography, will be discussed with reference to well-characterized systems.

Structure–function relationships in the hammerhead ribozyme probed by base rescue

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.

ATP binding to human serine racemase is cooperative and modulated by glycine.

The N‐methyl d‐aspartate (NMDA) receptors play a key role in excitatory neurotransmission, and control learning, memory and synaptic plasticity. Their activity is modulated by the agonist glutamate and by the co‐agonists d‐serine and glycine. In the human brain, d‐serine is synthesized from l‐serine by the dimeric pyridoxal 5′‐phosphate‐dependent enzyme serine racemase, which also degrades l‐ and d‐serine to pyruvate and ammonia. The dependence of l‐ and d‐serine β‐elimination and l‐serine racemization activities on ATP concentration was characterized, and was found to be strongly cooperative, with Hill coefficients close to 2 and apparent ATP dissociation constants ranging from 0.22 to 0.41 mm. ATP binding to the holo‐enzyme, monitored by the fluorescence changes of the coenzyme, was also determined to be cooperative, with an apparent dissociation constant of 0.24 mm. Glycine, an active‐site ligand, increased the serine racemase affinity for ATP by ~ 22‐fold, abolishing cooperativity. Conversely, ATP increased the non‐cooperative glycine binding15‐fold. These results indicate cross‐talk between allosteric and active sites, leading to the stabilization of two alternative protein conformations with ATP affinities of ~ 10 μM and 1.8 mm, as evaluated within the Monod, Wyman and Changeux model. Therefore, intracellular ATP and glycine control d‐serine homeostasis, and, indirectly, NMDA receptor activity. Because hyper‐ and hypo‐activation of NMDA receptors are associated with neuropathologies, the development of allosteric drugs modulating serine racemase activity is a promising therapeutic strategy.

The Advantages of Being Locked ASSESSING THE CLEAVAGE OF SHORT AND LONG RNAs BY LOCKED NUCLEIC ACID-CONTAINING 8–17 DEOXYRIBOZYMES

RNA-cleaving deoxyribozymes can be used for the sequence-specific knockdown of mRNAs. It was previously shown that activity of these deoxyribozymes is enhanced when their substrate-binding arms include some locked nucleic acid (LNA) residues, but the mechanistic basis of this enhancement was not explored. Here we dissected the kinetics and thermodynamics underlying the reaction of LNA-containing 8–17 deoxyribozymes. Four 8–17 constructs were designed to target sequences within the E6 mRNA from human papillomavirus type 16. When one of these deoxyribozymes (DNAzymes) and the corresponding LNA-armed enzyme (LNAzyme) were tested against a minimal RNA substrate, they showed similar rates of substrate binding and similar rates of intramolecular cleavage, but the LNAzyme released its substrate more slowly. The superior thermodynamic stability of the LNAzyme-substrate complex led to improved performances in reactions carried out at low catalyst concentrations. The four DNAzymes and the corresponding LNAzymes were then tested against extended E6 transcripts (>500 nucleotides long). With these structured substrates, the LNAzymes retained full activity, whereas the DNAzymes cleaved extremely poorly, unless they were allowed to pre-anneal to their targets. These results imply that LNAzymes can easily overcome the kinetic barrier represented by local RNA structure and bind to folded targets with a faster association rate as compared with DNAzymes. Such faster annealing to structured targets can be explained by a model whereby LNA monomers favor the initial hybridization to short stretches of unpaired residues (“nucleation”), which precedes disruption of the local mRNA structure and completion of the binding process.

An aminotransferase branch point connects purine catabolism to amino acid recycling

Although amino acids are known precursors of purines, a pathway for the direct recycling of amino acids from purines has never been described at the molecular level. We provide NMR and crystallographic evidence that the PucG protein from Bacillus subtilis catalyzes the transamination between an unstable intermediate ((S)-ureidoglycine) and the end product of purine catabolism (glyoxylate) to yield oxalurate and glycine. This activity enables soil and gut bacteria to use the animal purine waste as a source of carbon and nitrogen. The reaction catalyzed by (S)-ureidoglycine-glyoxylate aminotransferase (UGXT) illustrates a transamination sequence in which the same substrate provides both the amino group donor and, via its spontaneous decay, the amino group acceptor. Structural comparison and mutational analysis suggest a molecular rationale for the functional divergence between UGXT and peroxisomal alanine-glyoxylate aminotransferase, a fundamental enzyme for glyoxylate detoxification in humans.

Recombinant production of eight human cytosolic aminotransferases and assessment of their potential involvement in glyoxylate metabolism.

PH1 (primary hyperoxaluria type 1) is a severe inborn disorder of glyoxylate metabolism caused by a functional deficiency of the peroxisomal enzyme AGXT (alanine-glyoxylate aminotransferase), which converts glyoxylate into glycine using L-alanine as the amino-group donor. Even though pre-genomic studies indicate that other human transaminases can convert glyoxylate into glycine, in PH1 patients these enzymes are apparently unable to compensate for the lack of AGXT, perhaps due to their limited levels of expression, their localization in an inappropriate cell compartment or the scarcity of the required amino-group donor. In the present paper, we describe the cloning of eight human cytosolic aminotransferases, their recombinant expression as His6-tagged proteins and a comparative study on their ability to transaminate glyoxylate, using any standard amino acid as an amino-group donor. To selectively quantify the glycine formed, we have developed and validated an assay based on bacterial GO (glycine oxidase); this assay allows the detection of enzymes that produce glycine by transamination in the presence of mixtures of potential amino-group donors and without separation of the product from the substrates. We show that among the eight enzymes tested, only GPT (alanine transaminase) and PSAT1 (phosphoserine aminotransferase 1) can transaminate glyoxylate with good efficiency, using L-glutamate (and, for GPT, also L-alanine) as the best amino-group donor. These findings confirm that glyoxylate transamination can occur in the cytosol, in direct competition with the conversion of glyoxylate into oxalate. The potential implications for the treatment of primary hyperoxaluria are discussed.