New Insights about Enzyme Evolution from Large Scale Studies of Sequence and Structure Relationships

Understanding how enzymes have evolved offers clues about their structure-function relationships and mechanisms. Here, we describe evolution of functionally diverse enzyme superfamilies, each representing a large set of sequences that evolved from a common ancestor and that retain conserved features of their structures and active sites. Using several examples, we describe the different structural strategies nature has used to evolve new reaction and substrate specificities in each unique superfamily. The results provide insight about enzyme evolution that is not easily obtained from studies of one or only a few enzymes.     

Structural and mutagenesis studies on the cytochrome c peroxidase from Rhodobacter capsulatus provide new insights into structure-function relationships of bacterial di-heme peroxidases

Cytochrome c peroxidases (CCP) play a key role in cellular detoxification by catalyzing the reduction of hydrogen peroxide to water. The di-heme CCP from Rhodobacter capsulatus is the fastest enzyme (1060 s(-1)), when tested with its physiological cytochrome c substrate, among all di-heme CCPs characterized to date and has, therefore, been an attractive target to investigate structure-function relationships for this family of enzymes. Here, we combine for the first time structural studies with site-directed mutagenesis and spectroscopic studies of the mutant enzymes to investigate the roles of amino acid residues that have previously been suggested to be important for activity. The crystal structure of R. capsulatus at 2.7 Angstroms in the fully oxidized state confirms the overall molecular scaffold seen in other di-heme CCPs but further reveals that a segment of about 10 amino acids near the peroxide binding site is disordered in all four molecules in the asymmetric unit of the crystal. Structural and sequence comparisons with other structurally characterized CCPs suggest that flexibility in this part of the molecular scaffold is an inherent molecular property of the R. capsulatus CCP and of CCPs in general and that it correlates with the levels of activity seen in CCPs characterized, thus, far. Mutagenesis studies support the spin switch model and the roles that Met-118, Glu-117, and Trp-97 play in this model. Our results help to clarify a number of aspects of the debate on structure-function relationships in this family of bacterial CCPs and set the stage for future studies.     

Glutathione biosynthesis in bacteria by bifunctional GshF is driven by a modular structure featuring a novel hybrid ATP-grasp fold

Glutathione is an intracellular redox-active tripeptide thiol with a central role in cellular physiology across all kingdoms of life. Glutathione biosynthesis has been traditionally viewed as a conserved process relying on the sequential activity of two separate ligases, but recently, an enzyme (GshF) that unifies both necessary reactions in one platform has been identified and characterized in a number of pathogenic and free-living bacteria. Here, we report crystal structures of two prototypic GshF enzymes from Streptococcus agalactiae and Pasteurella multocida in an effort to shed light onto the structural determinants underlying their bifunctionality and to provide a structural framework for the plethora of biochemical and mutagenesis studies available for these enzymes. Our structures reveal how a canonical bacterial GshA module that catalyzes the condensation of L-glutamate and L-cysteine to γ-glutamylcysteine is linked to a novel ATP-grasp-like module responsible for the ensuing formation of glutathione from γ-glutamylcysteine and glycine. Notably, we identify an unprecedented subdomain in the ATP-grasp module of GshF at the interface of the GshF dimer, which is poised to mediate intersubunit communication and allosteric regulation of enzymatic activity. Comparison of the two GshF structures and mapping of structure-function relationships reveal that the bifunctional GshF structural platform operates as a dynamic dimeric assembly.     

Rieske business: structure-function of Rieske non-heme oxygenases

Rieske non-heme iron oxygenases (RO) catalyze stereo- and regiospecific reactions. Recently, an explosion of structural information on this class of enzymes has occurred in the literature. ROs are two/three component systems: a reductase component that obtains electrons from NAD(P)H, often a Rieske ferredoxin component that shuttles the electrons and an oxygenase component that performs catalysis. The oxygenase component structures have all shown to be of the alpha3 or alpha3beta3 types. The transfer of electrons happens from the Rieske center to the mononuclear iron of the neighboring subunit via a conserved aspartate, which is shown to be involved in gating electron transport. Molecular oxygen has been shown to bind side-on in naphthalene dioxygenase and a concerted mechanism of oxygen activation and hydroxylation of the ring has been proposed. The orientation of binding of the substrate to the enzyme is hypothesized to control the substrate selectivity and regio-specificity of product formation.     

Critical role of histidine residues in cyclohexanone monooxygenase expression, cofactor binding and catalysis

Cyclohexanone monooxygenase (CMO) is a member of the flavin monooxygenase superfamily of enzymes that catalyze both nucleophilic and electrophilic reactions involving a common C4a hydroperoxide intermediate. To begin to probe structure-function relationships for these enzymes, we investigated the roles of histidine residues in CMO derived from Acinetobacter NCIB 9871, with particular emphasis on the wholly conserved residue, His163 (H163). CMO activity was readily inactivated by diethyl pyrocarbonate (DEPC), a selective chemical modifier of histidine residues. Each of the seven histidines in CMO was then individually mutated to glutamine and the mutants expressed and purified from Escherichia coli. Only the H59Q mutant failed to express at significant levels. The H96Q enzyme was found to have a greatly reduced flavin adenine dinucleotide (FAD) content, indicative of compromised cofactor retention. The only significant effect on kcat occurred with the H163Q mutant, which exhibited an approximately 10-fold lower turnover of the prototypical substrate, cyclohexanone. This was accompanied by a doubling in the Km [NADPH] compared to the wild-type enzyme, suggesting that the functional decrement in H163Q is probably not solely a reflection of impaired NADPH binding. These data establish a critical role for H163 in CMO catalysis and prompt the hypothesis that this conserved residue plays a similarly important functional role across the flavin monooxygenase family of enzymes.     

Probing structure-function relationships and gating mechanisms in the CorA Mg2+ transport system

Recent crystal structures of the CorA Mg(2+) transport protein from Thermotoga maritima (TmCorA) revealed an unusually long ion pore putatively gated by hydrophobic residues near the intracellular end and by universally conserved asparagine residues at the periplasmic entrance. A conformational change observed in an isolated funnel domain structure also led to a proposal for the structural basis of gating. Because understanding the molecular mechanisms underlying ion channel and transporter gating remains an important challenge, we have undertaken a structure-guided engineering approach to probe structure-function relationships in TmCorA. The intracellular funnel domain is shown to constitute an allosteric regulatory module that can be engineered to promote an activated or closed state. A periplasmic gate centered about a proline-induced kink of the pore-lining helix is described where "helix-straightening" mutations produce a dramatic gain-of-function. Mutation to the narrowest constriction along the pore demonstrates that a hydrophobic gate is operational within this Mg(2+)-selective transport protein and likely forms an energetic barrier to ion flux. We also provide evidence that highly conserved acidic residues found in the short periplasmic loop are not essential for TmCorA function or Mg(2+) selectivity but may be required for proper protein folding and stability. This work extends our gating model for the CorA-Alr1-Mrs2 superfamily and reveals features that are characteristic of an ion channel. Aspects of these results that have broader implications for a range of channel and transporter families are highlighted.     

Progress in understanding structure-function relationships in respiratory chain complex II

Complex II (succinate:quinone oxidoreductase) of aerobic respiratory chains oxidizes succinate to fumarate and passes the electrons directly into the quinone pool. It serves as the only direct link between activity in the citric acid cycle and electron transport in the membrane. Finer details of these reactions and interactions are but poorly understood. However, complex II has extremely similar structural and catalytic properties to quinol:fumarate oxidoreductases of anaerobic organisms, for which X-ray structures have recently become available. These offer new insights into structure-function relationships of this class of flavoenzymes, including evidence favoring protein movement during catalysis.     

Glycomics: an integrated systems approach to structure-function relationships of glycans

In comparison with genomics and proteomics, the advancement of glycomics has faced unique challenges in the pursuit of developing analytical and biochemical tools and biological readouts to investigate glycan structure-function relationships. Glycans are more diverse in terms of chemical structure and information density than are DNA and proteins. This diversity arises from glycans' complex nontemplate-based biosynthesis, which involves several enzymes and isoforms of these enzymes. Consequently, glycans are expressed as an 'ensemble' of structures that mediate function. Moreover, unlike protein-protein interactions, which can be generally viewed as 'digital' in regulating function, glycan-protein interactions impinge on biological functions in a more 'analog' fashion that can in turn 'fine-tune' a biological response. This fine-tuning by glycans is achieved through the graded affinity, avidity and multivalency of their interactions. Given the importance of glycomics, this review focuses on areas of technologies and the importance of developing a bioinformatics platform to integrate the diverse datasets generated using the different technologies to allow a systems approach to glycan structure-function relationships.     

Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function

We performed alignment of apolipoprotein A-I (apoA-I) sequences from 31 species of animals. We found there is specific conservation of salt bridge-forming residues in the first 30 residues of apoA-I and general conservation of a variety of residue types in the central domain, helix 2/3 to helix 7/8. In the lipid-associating domain, helix 7 and helix 10 are the most and least conserved helixes, respectively. Furthermore, eight residues are completely conserved: P66, R83, P121, E191, and P220, and three of seven Tyr residues in human apoA-I, Y18, Y115, and Y192, are conserved. Residue Y18 appears to be important for assembly of HDL. E191-Y192 represents the only completely conserved pair of adjacent residues in apoA-I; Y192 is a preferred target for site-specific oxidative modification within atheroma, and molecular dynamic simulations suggest that the conserved pair E191-Y192 is in a solvent-exposed loop-helix-loop. Molecular dynamics testing of human apoA-I showed that M112 and M148 interact with Y115, a microenvironment unique to human apoA-I. Finally, conservation of Arg residues in the α11/3 helical wheel position 7 supports several possibilities: interactions with adjacent phospholipid molecules and/or oxidized lipids and/or binding of antioxidant enzymes through cation-π orbital interactions. We conclude that sequence alignment of apoA-I provides unique insights into apoA-I structure-function relationship.     

Phospholipases A2: Variations on a structural theme and their implications as to mechanism

Our approach to a study of structure-function relationships in phospholipases A₂ is based upon the hypothesis that these enzymes are related by divergent evolutionary processes. Accordingly, characterization of such enzymes, which exhibit structural and functional variations, must be reconciled with a common theme that ultimately relates to mechanism. Our focus in the present communication concerns studies on the bee venom enzyme and a new class of phospholipases A₂ that is unique in having a lysine in place of the aspartic acid at position 49.     

Accessing gap-junction channel structure-function relationships through molecular modeling and simulations

Background

Gap junction channels (GJCs) are massive protein channels connecting the cytoplasm of adjacent cells. These channels allow intercellular transfer of molecules up to ~1 kDa, including water, ions and other metabolites. Unveiling structure-function relationships coded into the molecular architecture of these channels is necessary to gain insight on their vast biological function including electrical synapse, inflammation, development and tissular homeostasis. From early works, computational methods have been critical to analyze and interpret experimental observations. Upon the availability of crystallographic structures, molecular modeling and simulations have become a valuable tool to assess structure-function relationships in GJCs. Modeling different connexin isoforms, simulating the transport process, and exploring molecular variants, have provided new hypotheses and out-of-the-box approaches to the study of these important channels.

Methods

Here, we review foundational structural studies and recent developments on GJCs using molecular modeling and simulation techniques, highlighting the methods and the cross-talk with experimental evidence.

Results and discussion

By comparing results obtained by molecular modeling and simulations techniques with structural and functional information obtained from both recent literature and structural databases, we provide a critical assesment of structure-function relationships that can be obtained from the junction between theoretical and experimental evidence.

     

Site-specific mutagenesis of PRD1 DNA polymerase: mutations in highly conserved regions of the family B DNA polymerase

The PRD1 DNA polymerase is a small multifunctional enzyme containing three major conserved amino acid sequences shared by family B DNA polymerases. Thus, the PRD1 DNA polymerase provides an useful model system with which to study structure-function relationships of DNA polymerase molecules. In order to investigate the functional and structural roles of the highly conserved amino acid sequences, we have introduced mutations into each of the 3 conserved regions of the PRD1 DNA polymerase. Genetic complementation study as well as DNA polymerase assay indicated that each mutation inactivated DNA polymerase catalytic activity, but not the 3' to 5' exonuclease activity.     

Amino acid conservation and clinical severity of human glucose-6-phosphate dehydrogenase mutations

More than a hundred naturally occurring mutations of human glucose-6-phosphate dehydrogenase (G6PD) have been identified at the amino acid level. The abundance of distinct mutation sites and their clinical manifestations make this enzyme ideal for structure-function analysis studies. We present here a sequence and structure combined analysis by which the severity of clinical symptoms resulting from point mutations of this enzyme is correlated with quantified degrees of amino acid conservation within 23 G6PD sequences from different organisms. Our analysis verifies, on a quantitative basis, a widely held notion that clinically severer mutations of G6PD usually occur at conserved amino acids. However, marked exceptions to this general trend exist which are most notably revealed by a number of mutations associated with chronic nonspherocytic hemolytic anemia (class I variants). When mapped onto a homology-derived structural model of human G6PD, these class I mutational sites of low amino acid conservation appear to localize in two spatially distinct clusters, both of which are populated with mutations consisting mainly of clinically severer variants (i.e. class I and class II). These results of computer-assisted analyses contribute to a further understanding of the structure-function relationships of human G6PD deficiency.     

Contribution of NMR spectroscopy to the study of structure-function relations of proteins and peptides

NMR spectroscopy provides a unique means to study molecular conformation, mechanisms of action and structure-function relationships for peptides and proteins in solution under conditions approaching those of their physiological environment. Development of NMR techniques, especially directed to the peptide and protein conformational analysis, is considered under the topics of two-level signal assignment and structural significance of homo- and heteronuclear spin-spin couplings. The results of NMR conformational analysis are presented for solution spatial structure of valinomy cin and gramicidin A antibiotics, honey-bee neurotoxin apamin, scorpion insectotoxin I5A and snake venom neurotoxins of "short" and "long" types. The structure-function relationships are discussed for these biologically active molecules.     

Human thyroid-stimulating hormone: structure-function analysis

This article provides the reader with an overview of methodological strategies to investigate structure-function relationships of human thyroid-stimulating hormone (hTSH). Various aspects of hTSH production, purification, and characterization described here in more detail are not only relevant to studies on other members of the glycoprotein hormone family, but also applicable to studies of other glycosylated proteins. Knowledge of structure-function relationships of specific hTSH domains is important for a better understanding of the molecular mechanisms of its action. New insights from such studies permit the design of glycoprotein hormone analogs with specific pharmacological properties and potential clinical applications.     

A single-molecule assay for telomerase structure-function analysis

The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures.     

Bicelles in structure-function studies of membrane-associated proteins

Bicelles are a novel form of long-chain/short-chain phospholipid aggregates, which are useful for biophysical and biochemical studies of membrane-associated biomolecules. In this work, we review the development of bicelles and their uses in structural characterization (primarily via NMR, circular dichroism, and fluorescence) of membrane-associated peptides. We also show that bicellar phospholipids are substrates for lipolytic enzymes. For this latter work, we employed a 31P NMR enzymatic assay system to examine the kinetic behavior of cobra venom phospholipase A(2) toward a variety of bicellar substrates. This enzyme hydrolyzed all bicelle lipids at rates comparable to those found for the enzyme action on traditional micellar substrates, which are the best substrates for this enzyme. In addition, we found that this PLA(2) showed no significant preference for long-chain or short-chain phospholipids when they were presented as mixtures in bicelles.     

Structure-Function Features of a Mycoplasma Glycolipid Synthase Derived from Structural Data Integration,Molecular Simulations,and Mutational Analysis

Glycoglycerolipids are structural components of mycoplasma membranes with a fundamental role in membrane properties and stability. Their biosynthesis is mediated by glycosyltransferases (GT) that catalyze the transfer of glycosyl units from a sugar nucleotide donor to diacylglycerol. The essential function of glycolipid synthases in mycoplasma viability, and the absence of glycoglycerolipids in animal host cells make these GT enzymes a target for drug discovery by designing specific inhibitors. However, rational drug design has been hampered by the lack of structural information for any mycoplasma GT. Most of the annotated GTs in pathogenic mycoplasmas belong to family GT2. We had previously shown that MG517 in Mycoplasma genitalium is a GT-A family GT2 membrane-associated glycolipid synthase. We present here a series of structural models of MG517 obtained by homology modeling following a multiple-template approach. The models have been validated by mutational analysis and refined by long scale molecular dynamics simulations. Based on the models, key structure-function relationships have been identified: The N-terminal GT domain has a GT-A topology that includes a non-conserved variable region involved in acceptor substrate binding. Glu193 is proposed as the catalytic base in the GT mechanism, and Asp40, Tyr126, Tyr169, Ile170 and Tyr218 define the substrates binding site. Mutation Y169F increases the enzyme activity and significantly alters the processivity (or sequential transferase activity) of the enzyme. This is the first structural model of a GT-A glycoglycerolipid synthase and provides preliminary insights into structure and function relationships in this family of enzymes.