Aminoglycoside Kinase Research Articles

Investigating Molecular Relationships Among Clinically Prevalent Aminoglycoside Phosphotransferases

Aminoglycosides constitute one of the most important class of antibiotics which are imperiled by the emergence of resistant organisms. Resistance to aminoglycosides is primarily due to enzymatic inactivation by several classes of aminoglycoside modifying enzymes including acetyl-transferases, nucleotidyl-transferases, and phospho-transferases. Among these Aminoglycoside phosphotransferases or kinases are an important class of antibiotic modifying enzymes which have been studied extensively. They are classified into seven different classes which include APH(2″)-I through IV, APH(3′)-I through VII, APH(3″)-I, APH(4)-I, APH(6)-I, APH(7″)-I APH(9)-I. Among these APH (2″) and APH (3′) groups are the largest and most prevalent clinically important enzymes. Several crystal structures of a number of APHs with diverse regiospecificity and substrate specificity have been elucidated. The present study investigates the molecular changes which facilitated the adaptation and mutational evolution of clinically prevalent APHs and the phylogenetic relationship using bioinformatics tools. Multiple sequence alignment of these enzymes using ClustalW reveals the diversity among the most conserved residues and mutational changes which are crucial for inactivation of the aminoglycoside. The mutational profile of the most critically conserved residues obtained can play a key role in understanding the molecular details of resistance mediated by most clinically prevalent APHs.

Molecular modeling studies to explore the binding affinity of virtually screened inhibitor toward different aminoglycoside kinases from diverse MDR strains.

Antibiotic resistance to aminoglycoside group of antibiotics mainly occurs by aminoglycoside kinases (AKs). Thus, targeting AKs from different multidrug resistant (MDR) strains could result into inhibition of AK enzymes and ultimately the resistance. Therefore, the present study aims to target one of these AKs that is APH(3')-Ia from Acinetobacter baumannii through structure based virtual screening (SBVS) and test the binding affinity of the most stable virtually screened inhibitor with AKs from Mycobacterium tuberculosis, Acinetobacter baumannii, Enterococcus gallinarum, and Escherichia coli. SBVS investigated ZINC71575479 as a most stable inhibitor with -8.92 kcal/mol of binding energy and 0.66 µM of inhibition constant. Molecular docking results revealed that the ZINC71575479 can efficiently bind to nucleotide triphosphate (NTP) binding site of different AKs which is a known drug target site. Sequence analysis study of different AKs showed no significant similarity for active site residues; however structure superimposition study showed conserved NTP-binding domain. Molecular dynamics (MD) simulations showed stable behavior of all docked complexes with notable conformational stability of salt bridges at NTP-binding site of different AKs. Binding energy calculations revealed the interactions between key residues from NTP- binding domain of different AKs with ZINC71575479. In order to validate the MD simulations and binding energy results, crystal structure complexed with tyrphostin AG1478 a known inhibitor of AKs was kept as control. Thus, this work demonstrates the binding efficiency of ZINC71575479 toward different AKs for circumventing aminoglycoside resistance and opens avenues for the development of new antibiotics that can target diverse MDR strains with aminoglycoside resistance.

Antibiotic Binding Drives Catalytic Activation of Aminoglycoside Kinase APH(2″)-Ia

APH(2″)-Ia is a widely disseminated resistance factor frequently found in clinical isolates of Staphylococcus aureus and pathogenic enterococci, where it is constitutively expressed. APH(2″)-Ia confers high-level resistance to gentamicin and related aminoglycosides through phosphorylation of the antibiotic using guanosine triphosphate (GTP) as phosphate donor. We have determined crystal structures of the APH(2″)-Ia in complex with GTP analogs, guanosine diphosphate, and aminoglycosides. These structures collectively demonstrate that aminoglycoside binding to the GTP-bound kinase drives conformational changes that bring distant regions of the protein into contact. These changes in turn drive a switch of the triphosphate cofactor from an inactive, stabilized conformation to a catalytically competent active conformation. This switch has not been previously reported for antibiotic kinases or for the structurally related eukaryotic protein kinases. This catalytic triphosphate switch presents a means by which the enzyme can curtail wasteful hydrolysis of GTP in the absence of aminoglycosides, providing an evolutionary advantage to this enzyme.

Identification and classification of small molecule kinases: insights into substrate recognition and specificity.

BackgroundMany prokaryotic kinases that phosphorylate small molecule substrates, such as antibiotics, lipids and sugars, are evolutionarily related to Eukaryotic Protein Kinases (EPKs). These Eukaryotic-Like Kinases (ELKs) share the same overall structural fold as EPKs, but differ in their modes of regulation, substrate recognition and specificity—the sequence and structural determinants of which are poorly understood.ResultsTo better understand the basis for ELK specificity, we applied a Bayesian classification procedure designed to identify sequence determinants responsible for functional divergence. This reveals that a large and diverse family of aminoglycoside kinases, characterized members of which are involved in antibiotic resistance, fall into major sub-groups based on differences in putative substrate recognition motifs. Aminoglycoside kinase substrate specificity follows simple rules of alternating hydroxyl and amino groups that is strongly correlated with variations at the DFG + 1 position.ConclusionsSubstrate specificity determining features in small molecule kinases are mostly confined to the catalytic core and can be identified based on quantitative sequence and crystal structure comparisons.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0576-x) contains supplementary material, which is available to authorized users.

Determinants of nucleoside specificity of a macrolide phosphotransferase

2'-macrolide phosphotransferase type I [MPH(2')-I] is an antibiotic kinase that renders many macrolides, such as erythromycin, inactive by catalyzing the transfer of a phosphate group from a nucleoside triphosphate to the hydroxyl at the 2'-position of the antibiotic. MPH(2')-I is functionally and structurally analogous to the aminoglycoside kinases (APHs). However, it is distinct from most APHs in that it utilizes GTP exclusively as its phosphate donor. We will present the crystal structure of MPH(2')-I in its apo and ternary complex forms with guanosine nucleotide and different macrolide substrates. We will compare its nucleoside-binding pocket to that of the 2''-aminoglycoside phosphotransferases [APH(2'')], a subclass of aminoglycoside kinases that are capable of utilizing GTP as a phosphate donor. To further decipher the structural basis of the nucleoside specificity of MPH(2')-I, mutations of amino acid resides in the nucleoside-binding pocket have been carried out and their effects on the binding affinity of purine nucleotides were examined by isothermal titration calorimetry. Our preliminary results show that the "gatekeeper" residue plays a role in governing the nucleoside selectivity.

Antibiotic resistance and the golden age of microbiology

Antibiotic resistance and the golden age of microbiology

Simultaneous Inhibition Assay for Human and Microbial Kinases via MALDI‐MS/MS

Selective inhibition of one kinase over another is a critical issue in drug development. For antimicrobial development, it is particularly important to selectively inhibit bacterial kinases, which can phosphorylate antimicrobial compounds such as aminoglycosides, without affecting human kinases. Previous work from our group showed the development of a MALDI-MS/MS assay for the detection of small molecule modulators of the bacterial aminoglycoside kinase APH3'IIIa. Herein, we demonstrate the development of an enhanced kinase MALDI-MS/MS assay involving simultaneous assaying of two kinase reactions, one for APH3'IIIa, and the other for human protein kinase A (PKA), which leads to an output that provides direct information on selectivity and mechanism of action. Specificity of the respective enzyme substrates were verified, and the assay was validated through generation of Z'-factors of 0.55 for APH3'IIIa with kanamycin and 0.60 for PKA with kemptide. The assay was used to simultaneously screen a kinase-directed library of mixtures of ten compounds each against both enzymes, leading to the identification of selective inhibitors for each enzyme as well as one non-selective inhibitor following mixture deconvolution.

A matrix-assisted laser desorption/ionization tandem mass spectrometry method for direct screening of small molecule mixtures against an aminoglycoside kinase

Aminoglycoside phosphotransferase 3′IIIa (APH3′IIIa) is a bacterial enzyme involved in antibiotic resistance through phosphorylation of aminoglycosides, which can potentially be overcome by co-administration of an APH3′IIIa inhibitor with the antibiotic. Current assay methods for discovery of APH3′IIIa inhibitors suffer from low specificity and high false positive/negative hit rates. Here, we describe a method for screening APH3′IIIa inhibitors based on direct detection of kanamycin A phosphorylation using MALDI-MS/MS, which is more rapid than conventional assays and does not require secondary assays or sample cleanup. The MALDI-MS/MS assay operates at an ionic strength of 45mM and co-factors can be utilized at near-physiological levels for optimal enzyme activity. Detection via MALDI-MS/MS allowed for improved reproducibility when compared to ESI-MS/MS. Furthermore, the use of MS/MS provided better signal-to-noise ratios relative to MS alone on the MALDI instrument. The assay was validated via generation of Z′-factors, with values of 0.78 and 0.56 in the absence and presence of 0.2% DMSO, respectively. The assay was used to screen a kinase directed library of >200 compounds, assayed as 21 mixtures of 10 compounds each. Five novel synthetic inhibitors were identified following mixture deconvolution. Inhibition constants were obtained for the aforementioned inhibitors using the MALDI-MS/MS assay, revealing several low to mid micromolar “hits”, and highlighting the quantitative nature of the assay.

Thermodynamical properties of protein kinase with adenine inhibitors

The protein-based molecular recognition of the adenine ring is essential to understand protein function and drug design as well. In this paper, a variety of the adenine-based inhibitors with modified groups of amino groups, nitrogen and oxygen atoms in the aromatic ring are designed, and the binding capability of these adenosine analogues with an aminoglycoside antibiotic kinase [APH(3′)-IIIa] are investigated with activity assays and isothermal titration calorimetry (ITC) experiments. 1-aminoisoquinoline is one of the weakest substrates bound to APH(3′)-IIIa with the lowest affinity (high ki and high kd) and the smallest negative value of free energy change (ΔG) among the inhibitors tested. The binding process of adenine and 5-nitroisoquinoline to APH(3′)-IIIa is an enthalpy-driven event with unfavorable entropy, which is consistent with the energy change induced by the binding of ATP to the enzyme. However, the reverse is true for 1-aminoisoquinoline, 3-amino-5-nitrobenzisothiazole, 5-aminoisoquinoline binding to the enzyme because the entropy is more favorable and the enthalpy makes a lower contribution to the binding process. These results suggest that small changes of the adenine ring can lead to significant influence on the ability of these analogues to occupy the adenine-binding region of the enzyme, which can be the potential inhibitors as drug candidates against the bacterial resistance.

Prospects for circumventing aminoglycoside kinase mediated antibiotic resistance.

Aminoglycosides are a class of antibiotics with a broad spectrum of antimicrobial activity. Unfortunately, resistance in clinical isolates is pervasive, rendering many aminoglycosides ineffective. The most widely disseminated means of resistance to this class of antibiotics is inactivation of the drug by aminoglycoside-modifying enzymes (AMEs). There are two principal strategies to overcoming the effects of AMEs. The first approach involves the design of novel aminoglycosides that can evade modification. Although this strategy has yielded a number of superior aminoglycoside variants, their efficacy cannot be sustained in the long term. The second approach entails the development of molecules that interfere with the mechanism of AMEs such that the activity of aminoglycosides is preserved. Although such a molecule has yet to enter clinical development, the search for AME inhibitors has been greatly facilitated by the wealth of structural information amassed in recent years. In particular, aminoglycoside phosphotransferases or kinases (APHs) have been studied extensively and crystal structures of a number of APHs with diverse regiospecificity and substrate specificity have been elucidated. In this review, we present a comprehensive overview of the available APH structures and recent progress in APH inhibitor development, with a focus on the structure-guided strategies.

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6')-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6')-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k(cat)/K(m) = 10(5)-10(7) M(-1) s(-1)). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.

Aminoglycoside 2′′-Phosphotransferase IIIa (APH(2′′)-IIIa) Prefers GTP over ATP: STRUCTURAL TEMPLATES FOR NUCLEOTIDE RECOGNITION IN THE BACTERIAL AMINOGLYCOSIDE-2′′ KINASES

Contrary to the accepted dogma that ATP is the canonical phosphate donor in aminoglycoside kinases and protein kinases, it was recently demonstrated that all members of the bacterial aminoglycoside 2''-phosphotransferase IIIa (APH(2'')) aminoglycoside kinase family are unique in their ability to utilize GTP as a cofactor for antibiotic modification. Here we describe the structural determinants for GTP recognition in these enzymes. The crystal structure of the GTP-dependent APH(2'')-IIIa shows that although this enzyme has templates for both ATP and GTP binding superimposed on a single nucleotide specificity motif, access to the ATP-binding template is blocked by a bulky tyrosine residue. Substitution of this tyrosine by a smaller amino acid opens access to the ATP template. Similar GTP binding templates are conserved in other bacterial aminoglycoside kinases, whereas in the structurally related eukaryotic protein kinases this template is less conserved. The aminoglycoside kinases are important antibiotic resistance enzymes in bacteria, whose wide dissemination severely limits available therapeutic options, and the GTP binding templates could be exploited as new, previously unexplored targets for inhibitors of these clinically important enzymes.

Structural Basis for Dual Nucleotide Selectivity of Aminoglycoside 2″-Phosphotransferase IVa Provides Insight on Determinants of Nucleotide Specificity of Aminoglycoside Kinases

Enzymatic phosphorylation through a family of enzymes called aminoglycoside O-phosphotransferases (APHs) is a major mechanism by which bacteria confer resistance to aminoglycoside antibiotics. Members of the APH(2″) subfamily are of particular clinical interest because of their prevalence in pathogenic strains and their broad substrate spectra. APH(2″) enzymes display differential preferences between ATP or GTP as the phosphate donor, with aminoglycoside 2″-phosphotransferase IVa (APH(2″)-IVa) being a member that utilizes both nucleotides at comparable efficiencies. We report here four crystal structures of APH(2″)-IVa, two of the wild type enzyme and two of single amino acid mutants, each in complex with either adenosine or guanosine. Together, these structures afford a detailed look at the nucleoside-binding site architecture for this enzyme and reveal key elements that confer dual nucleotide specificity, including a solvent network in the interior of the nucleoside-binding pocket and the conformation of an interdomain linker loop. Steady state kinetic studies, as well as sequence and structural comparisons with members of the APH(2″) subfamily and other aminoglycoside kinases, rationalize the different substrate preferences for these enzymes. Finally, despite poor overall sequence similarity and structural homology, analysis of the nucleoside-binding pocket of APH(2″)-IVa shows a striking resemblance to that of eukaryotic casein kinase 2 (CK2), which also exhibits dual nucleotide specificity. These results, in complement with the multitude of existing inhibitors against CK2, can serve as a structural basis for the design of nucleotide-competitive inhibitors against clinically relevant APH enzymes.

A Small Molecule Discrimination Map of the Antibiotic Resistance Kinome

Kinase-mediated resistance to antibiotics is a significant clinical challenge. These enzymes sharea common protein fold characteristic of Ser/Thr/Tyr protein kinases. We screened 14 antibiotic resistance kinases against 80 chemically diverse protein kinase inhibitors to map resistance kinase chemical space. The screens identified molecules with both broad and narrow inhibition profiles, proving that protein kinase inhibitors offer privileged chemical matter with the potential to block antibiotic resistance. One example is the flavonol quercetin, which inhibited a number of resistance kinases invitro and invivo. This activity was rationalized by determination of the crystal structure of the aminoglycoside kinase APH(2″)-IVa in complex with quercetin and its antibiotic substrate kanamycin. Our data demonstrate that protein kinase inhibitors offer chemical scaffolds that can block antibiotic resistance, providing leads for co-drug design.

Crystal Structures of Two Aminoglycoside Kinases Bound with a Eukaryotic Protein Kinase Inhibitor

Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3′)-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3′)-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eurkaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides.

Mapping of the ATP-binding domain of human fructosamine 3-kinase-related protein by affinity labelling with 5′-[p-(fluorosulfonyl)benzoyl]adenosine

The modification of proteins by reducing sugars through the process of non-enzymatic glycation is one of the principal mechanisms by which hyperglycaemia may precipitate the development of diabetic complications. Fn3K (fructosamine 3-kinase) and Fn3KRP (Fn3K-related protein) are two recently discovered enzymes that may play roles in metabolizing early glycation products. However, although the activity of these enzymes towards various glycated substrates has been established, very little is known about their structure-function relationships or their respective mechanisms of action. Furthermore, their only structural similarities noted to date with members of other kinase families has been with the bacterial aminoglycoside kinases. In the present study, we employed affinity labelling with the ATP analogue FSBA {5'-p-[(fluorosulfonyl)benzoyl]adenosine} to probe the active-site topology of Fn3KRP as an example of this enigmatic family of kinases. FSBA was found to modify Fn3KRP at five distinct sites; four of these were predicted to be localized in close proximity to its ATP-binding site, based on alignments with the aminoglycoside kinase APH(3')-IIIa, and examination of its published tertiary structure. The results of the present studies provide evidence that Fn3KRP possesses an ATP-binding domain that is structurally related to that of both the aminoglycoside kinases and eukaryotic protein kinases.

Structural Evolution of the Protein Kinase–Like Superfamily

The protein kinase family is large and important, but it is only one family in a larger superfamily of homologous kinases that phosphorylate a variety of substrates and play important roles in all three superkingdoms of life. We used a carefully constructed structural alignment of selected kinases as the basis for a study of the structural evolution of the protein kinase–like superfamily. The comparison of structures revealed a “universal core” domain consisting only of regions required for ATP binding and the phosphotransfer reaction. Remarkably, even within the universal core some kinase structures display notable changes, while still retaining essential activity. Hence, the protein kinase–like superfamily has undergone substantial structural and sequence revision over long evolutionary timescales. We constructed a phylogenetic tree for the superfamily using a novel approach that allowed for the combination of sequence and structure information into a unified quantitative analysis. When considered against the backdrop of species distribution and other metrics, our tree provides a compelling scenario for the development of the various kinase families from a shared common ancestor. We propose that most of the so-called “atypical kinases” are not intermittently derived from protein kinases, but rather diverged early in evolution to form a distinct phyletic group. Within the atypical kinases, the aminoglycoside and choline kinase families appear to share the closest relationship. These two families in turn appear to be the most closely related to the protein kinase family. In addition, our analysis suggests that the actin-fragmin kinase, an atypical protein kinase, is more closely related to the phosphoinositide-3 kinase family than to the protein kinase family. The two most divergent families, α-kinases and phosphatidylinositol phosphate kinases (PIPKs), appear to have distinct evolutionary histories. While the PIPKs probably have an evolutionary relationship with the rest of the kinase superfamily, the relationship appears to be very distant (and perhaps indirect). Conversely, the α-kinases appear to be an exception to the scenario of early divergence for the atypical kinases: they apparently arose relatively recently in eukaryotes. We present possible scenarios for the derivation of the α-kinases from an extant kinase fold.

Establishing the Principles of Recognition in the Adenine-Binding Region of an Aminoglycoside Antibiotic Kinase [APH(3‘)-IIIa

The protein-based molecular recognition of the adenine ring has implications throughout biological systems. In this paper, we discuss the adenine-binding region of an aminoglycoside antibiotic kinase [APH(3')-IIIa], which serves as an excellent model system for proteins that bind the adenine ring. This enzyme employs a hydrogen-bonding network involving water molecules along with enzyme backbone/side-chain atoms and a pi-pi stacking interaction to recognize the adenine ring. Our approach utilized site-directed mutagenesis, adenosine analogues and a variety of biophysical methods to probe the contacts in the adenine-binding region of APH(3')-IIIa. The results point to the polar nature of an adenine-Met90 contact in this binding pocket and the important role that Met90, the "gatekeeper" residue in structurally similar Ser/Thr protein kinases, plays in adenine binding. The results also suggest that small changes in the structure of the adenine ring can lead to significant changes in the ability of these analogues to occupy the adenine-binding region of the enzyme. Additional computational experiments indicate that both size and electronic factors are important in the binding of aromatic systems in this interaction-rich pocket. The principles governing adenine recognition established in this study may be applied to other protein-ligand complexes and used to navigate future studies directed at discovering potent and selective inhibitors of APH-type enzymes.

A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases

Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.

Structural Evolution of the Protein Kinase-Like Superfamily

The protein kinase family is large and important, but it is only one family in a larger superfamily of homologous kinases that phosphorylate a variety of substrates and play important roles in all three superkingdoms of life. We used a carefully constructed structural alignment of selected kinases as the basis for a study of the structural evolution of the protein kinase–like superfamily. The comparison of structures revealed a “universal core” domain consisting only of regions required for ATP binding and the phosphotransfer reaction. Remarkably, even within the universal core some kinase structures display notable changes, while still retaining essential activity. Hence, the protein kinase–like superfamily has undergone substantial structural and sequence revision over long evolutionary timescales. We constructed a phylogenetic tree for the superfamily using a novel approach that allowed for the combination of sequence and structure information into a unified quantitative analysis. When considered against the backdrop of species distribution and other metrics, our tree provides a compelling scenario for the development of the various kinase families from a shared common ancestor. We propose that most of the so-called “atypical kinases” are not intermittently derived from protein kinases, but rather diverged early in evolution to form a distinct phyletic group. Within the atypical kinases, the aminoglycoside and choline kinase families appear to share the closest relationship. These two families in turn appear to be the most closely related to the protein kinase family. In addition, our analysis suggests that the actin-fragmin kinase, an atypical protein kinase, is more closely related to the phosphoinositide-3 kinase family than to the protein kinase family. The two most divergent families, α-kinases and phosphatidylinositol phosphate kinases (PIPKs), appear to have distinct evolutionary histories. While the PIPKs probably have an evolutionary relationship with the rest of the kinase superfamily, the relationship appears to be very distant (and perhaps indirect). Conversely, the α-kinases appear to be an exception to the scenario of early divergence for the atypical kinases: they apparently arose relatively recently in eukaryotes. We present possible scenarios for the derivation of the α-kinases from an extant kinase fold.