Conservation of caspase substrates across metazoans suggests hierarchical importance of signaling pathways over specific targets and cleavage site motifs in apoptosis.

Caspases, cysteine proteases with aspartate specificity, are key players in programmed cell death across the metazoan lineage. Hundreds of apoptotic caspase substrates have been identified in human cells. Some have been extensively characterized, revealing key functional nodes for apoptosis signaling and important drug targets in cancer. But the functional significance of most cuts remains mysterious. We set out to better understand the importance of caspase cleavage specificity in apoptosis by asking which cleavage events are conserved across metazoan model species. Using N-terminal labeling followed by mass spectrometry, we identified 257 caspase cleavage sites in mouse, 130 in Drosophila, and 50 in Caenorhabditis elegans. The large majority of the caspase cut sites identified in mouse proteins were found conserved in human orthologs. However, while many of the same proteins targeted in the more distantly related species were cleaved in human orthologs, the exact sites were often different. Furthermore, similar functional pathways are targeted by caspases in all four species. Our data suggest a model for the evolution of apoptotic caspase specificity that highlights the hierarchical importance of functional pathways over specific proteins, and proteins over their specific cleavage site motifs.

Divergent evolution of enzymatic function: mechanistically diverse superfamilies and functionally distinct suprafamilies.

The protein sequence and structure databases are now sufficiently representative that strategies nature uses to evolve new catalytic functions can be identified. Groups of divergently related enzymes whose members catalyze different reactions but share a common partial reaction, intermediate, or transition state (mechanistically diverse superfamilies) have been discovered, including the enolase, amidohydrolase, thiyl radical, crotonase, vicinal-oxygen-chelate, and Fe-dependent oxidase superfamilies. Other groups of divergently related enzymes whose members catalyze different overall reactions that do not share a common mechanistic strategy (functionally distinct suprafamilies) have also been identified: (a) functionally distinct suprafamilies whose members catalyze successive transformations in the tryptophan and histidine biosynthetic pathways and (b) functionally distinct suprafamilies whose members catalyze different reactions in different metabolic pathways. An understanding of the structural bases for the catalytic diversity observed in super- and suprafamilies may provide the basis for discovering the functions of proteins and enzymes in new genomes as well as provide guidance for in vitro evolution/engineering of new enzymes.

Functional assignment of the 20 S proteasome from Trypanosoma brucei using mass spectrometry and new bioinformatics approaches.

As experimental technologies for characterization of proteomes emerge, bioinformatic analysis of the data becomes essential. Separation and identification technologies currently based on two-dimensional gels/mass spectrometry provide the inherent analytical power required. This strategy involves protein spot digestion and accurate mass mapping together with computational interrogation of available data bases for protein functional identification. When either no exact match is found or when the possible matches only partially account for molecular weights actually observed, peptide sequencing by tandem mass spectrometry has emerged as the methodology of choice to provide the basic additional information required. To evaluate the capabilities of bioinformatics methods employed for identifying homologs of a protein of interest, we attempted to identify the major proteins from the 20 S proteasome of Trypanosoma brucei using sequence information determined using mass spectrometry. The results suggest that neither the traditional query engines, BLAST and FASTA, nor specialized software developed for analysis of sequence information obtained by mass spectrometry are able to identify even closely related sequences at statistically significant scores. To address this deficit, new bioinformatics approaches were developed for concomitant use of the multiple fragments of short sequence typically available from methods of tandem mass spectrometry. These approaches rely on the occurrence of congruence across searches of multiple fragments from a single protein. This method resulted in sharply better statistical significance values for correct hits in the data base output relative to that achieved for independent searches using single sequence fragments.

Mutagenesis of two acidic active site residues in human muscle creatine kinase: implications for the catalytic mechanism.

Creatine kinase (CK) catalyzes the reversible phosphorylation of the guanidine substrate, creatine, by MgATP. Although several X-ray crystal structures of various isoforms of creatine kinase have been published, the detailed catalytic mechanism remains unresolved. A crystal structure of the CK homologue, arginine kinase (AK), complexed with the transition-state analogue (arginine-nitrate-ADP), has revealed two carboxylate amino acid residues (Glu225 and Glu314) within 2.8 A of the proposed transphosphorylation site. These two residues are the putative catalytic groups that may promote nucleophilic attack by the guanidine amino group on the gamma-phosphate of ATP. From primary sequence alignments of arginine kinases and creatine kinases, we have identified two homologous creatine kinase acidic amino acid residues (Glu232 and Asp326), and these were targeted for examination of their potential roles in the CK mechanism. Using site-directed mutagenesis, we have made several substitutions at these two positions. The results indicate that of these two residues the Glu232 is the likely catalytic residue while Asp326 likely performs a role in properly aligning substrates for catalysis.

ViewFeature: integrated feature analysis and visualization.

Visualization interfaces for high performance computing systems pose special problems due to the complexity and volume of data these systems manipulate. In the post-genomic era, scientists must be able to quickly gain insight into structure-function problems, and require flexible computing environments to quickly create interfaces that link the relevant tools. Feature, a program for analyzing protein sites, takes a set of 3-dimensional structures and creates statistical models of sites of structural or functional significance. Until now, Feature has provided no support for visualization, which can make understanding its results difficult. We have developed an extension to the molecular visualization program Chimera that integrates Feature's statistical models and site predictions with 3-dimensional structures viewed in Chimera. We call this extension ViewFeature, and it is designed to help users understand the structural Features that define a site of interest. We applied ViewFeature in an analysis of the enolase superfamily; a functionally distinct class of proteins that share a common fold, the alpha/beta barrel, in order to gain a more complete understanding of the conserved physical properties of this superfamily. In particular, we wanted to define the structural determinants that distinguish the enolase superfamily active site scaffold from other alpha/beta barrel superfamilies and particularly from other metal-binding alpha/beta barrel proteins. Through the use of ViewFeature, we have found that the C-terminal domain of the enolase superfamily does not differ at the scaffold level from metal-binding alpha/beta barrels. We are, however, able to differentiate between the metal-binding sites of alpha/beta barrels and those of other metal-binding proteins. We describe the overall architectural Features of enolases in a radius of 10 Angstroms around the active site.

A comparative study of human muscle and brain creatine kinases expressed in Escherichia coli.

We report the expression of the human muscle (CK-MM) and brain (CK-BB) creatine kinases in Escherichia coli. The proteins have been purified to apparent homogeneity and several of their physical and kinetic properties investigated. In the process, we have conclusively verified the correct DNA sequence of the genes encoding the respective isozymes, and determined the correct primary structure and mass of the gene products. Alignment of the primary sequences of these two enzymes shows 81% sequence identity with each other, and no obvious gross structural differences. However, Western blot analyses demonstrated the general lack of antigenic cross-reactivity between these isozymes. Preliminary kinetic analyses show the K(m) and k(cat) values for the creatine and MgATP substrates are similar to values reported for other isozymes from various tissues and organisms. The human muscle and brain CKs do not, however, exhibit the synergism of substrate binding that is observed, for example, in rabbit muscle creatine kinase.

Integrated tools for structural and sequence alignment and analysis.

We have developed new computational methods for displaying and analyzing members of protein superfamilies. These methods (MinRMS, AlignPlot and MSFviewer) integrate sequence and structural information and are implemented as separate but cooperating programs to our Chimera molecular modeling system. Integration of multiple sequence alignment information and three-dimensional structural representations enable researchers to generate hypotheses about the sequence-structure relationship. Structural superpositions can be generated and easily tuned to identify similarities around important characteristics such as active sites or ligand binding sites. Information related to the release of Chimera, MinRMS, AlignPlot and MSFviewer can be obtained at http:¿www.cgl.ucsf.edu/chimera.

Can sequence determine function?

The functional annotation of proteins identified in genome sequencing projects is based on similarities to homologs in the databases. As a result of the possible strategies for divergent evolution, homologous enzymes frequently do not catalyze the same reaction, and we conclude that assignment of function from sequence information alone should be viewed with some skepticism.

Shotgun: getting more from sequence similarity searches.

MOTIVATION: As genomic sequencing reveals the range of structural classes generated through the evolution of proteins, analysis of the superfamilies to which they belong can contribute important insights for understanding their structure-function relationships. Current database search techniques fall short of identifying the majority of distant sequence relationships at statistically significant levels. We developed the Shotgun program in an effort to enhance the sensitivity and utility of current database search output. RESULTS: We have developed and used the Shotgun program to identify both new superfamily members and to reconstruct several known enzyme superfamilies using BLAST database searches. An analysis of the false-positive rates generated in the analysis and other control experiments provides evidence that high Shotgun scores indicate real evolutionary relationships. Shotgun is also a useful tool for identifying subgroup relationships within superfamilies and for testing hypotheses about related protein families. AVAILABILITY: By request from the Babbitt lab homepage: http://mako.cgl.ucsf. edu/babbittlab/ CONTACT: babbitt@cgl.ucsf.edu

Evidence that pcpA encodes 2,6-dichlorohydroquinone dioxygenase, the ring cleavage enzyme required for pentachlorophenol degradation in Sphingomonas chlorophenolica strain ATCC 39723.

An enzyme that catalyzes an Fe2+-dependent reaction of 2, 6-dichlorohydroquinone with O2 has been isolated from Sphingomonas chlorophenolica sp. strain ATCC 39723, a soil microorganism capable of complete mineralization of pentachlorophenol. The product of the reaction is too unstable to allow spectroscopic characterization, but is apparently negatively charged and retains the two chlorine atoms of the substrate. The enzyme was partially sequenced using electrospray LC-MS, and one peptide was used to search the NCBInr database. This peptide matched a part of PcpA, a protein of unknown function that is induced in S. chlorophenolica in response to pentachlorophenol. Several other peptides could also be mapped onto the sequence of PcpA, suggesting that the enzyme is encoded by pcpA. PcpA has low but significant sequence similarity to an unusual class of extradiol dioxygenases. On the basis of the sequence analysis, the Fe2+ and O2 dependence of the enzyme, and the characteristics of the product, the enzyme is proposed to be a 2,6-dichlorohydroquinone dioxygenase. The position of ring cleavage has not yet been identified.

Unexpected divergence of enzyme function and sequence: "N-acylamino acid racemase" is o-succinylbenzoate synthase.

A protein identified as "N-acylamino acid racemase" from Amycolaptosis sp. is an inefficient enzyme (kcat/Km = 3.7 x 10(2) M-1 s-1). Its sequence is 43% identical to that of an unidentified protein encoded by the Bacillus subtilis genome. Both proteins efficiently catalyze the o-succinylbenzoate synthase reaction in menaquinone biosynthesis (kcat/Km = 2.5 x 10(5) and 7.5 x 10(5) M-1 s-1, respectively), suggesting that this is their "correct" metabolic function. Their membership in the mechanistically diverse enolase superfamily provides an explanation for the catalytic promiscuity of the protein from Amycolaptosis. The adventitious promiscuity may provide an example of a protein poised for evolution of a new enzymatic function in the enolase superfamily. This study demonstrates that the correct assignment of function to new proteins in functional and structural genomics may require an understanding of the metabolism of the organism.

Mechanistically diverse enzyme superfamilies: the importance of chemistry in the evolution of catalysis.

The strategy that nature has used to evolve new catalytic activities from pre-existing enzymes (i.e. retention of substrate binding or of catalytic mechanism) has been controversial. Recent work supports a strategy in which a partial reaction, catalyzed by a progenitor, is retained, and the active-site architecture is modified to allow the intermediate generated to be directed to different products.

Evolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida.

The structure of (D)-glucarate dehydratase from Pseudomonas putida (GlucD) has been solved at 2.3 A resolution by multiple isomorphous replacement and refined to a final R-factor of 19.0%. The protein crystallizes in the space group I222 with one subunit in the asymmetric unit. The unit cell dimensions are a = 69.6 A, b = 108.8 A, and c = 122.6 A. The crystals were grown using the batch method where the primary precipitant was poly(ethylene glycol) 1000. The structure reveals that GlucD is a tetramer of four identical polypeptides, each containing 451 residues. The structure was determined without a bound substrate or substrate analogue. Three disordered regions are noted: the N-terminus through residue 11, a loop containing residues 99 through 110, and the C-terminus from residue 423. On the basis of primary sequence alignments, we previously concluded that GlucD is a member of the mandelate racemase (MR) subfamily of the enolase superfamily [Babbitt, P. C., Hasson, M. S., Wedekind, J. E., Palmer, D. R. J., Barrett, W. C., Reed, G. J., Rayment, I., Ringe, D., Kenyon, G. L., and Gerlt, J. A. (1996) Biochemistry 35, 16489-16501]. This prediction is now verified, since the overall fold of GlucD is strikingly similar to those of MR, muconate lactonizing enzyme I, and enolase. Also, many of the active site residues of GlucD can be superimposed on those found in the active site of MR. The implications of this structure on the evolution of catalysis in the enolase superfamily are discussed.

Evolution of enzymatic activities in the enolase superfamily: characterization of the (D)-glucarate/galactarate catabolic pathway in Escherichia coli.

The genes encoding the enzymes in the (D)-glucarate/galactarate catabolic pathway have been identified in the Escherichia coli genome. These encode, in three transcriptional units, (D)-glucarate dehydratase (GlucD), galactarate dehydratase, 5-keto-4-deoxy-(D)-glucarate aldolase, tartronate semialdehyde reductase, a glycerate kinase that generates 2-phosphoglycerate as product, and two hexaric acid transporters. We also have identified a gene proximal to that encoding GlucD that encodes a protein that is 72% identical in primary sequence to GlucD (GlucD-related protein or GlucDRP). However, whereas GlucD catalyzes the efficient dehydration of both (D)-glucarate and (L)-idarate as well as their epimerization, GlucDRP is significantly impaired in both reactions. Perhaps GlucDRP is an example of gene duplication and evolution in progress in the E. coli chromosome.

Evolution of an enzyme active site: the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase.

Muconate lactonizing enzyme (MLE), a component of the beta-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enzymes (the "enolase superfamily") that catalyze the abstraction of the alpha-proton of a carboxylic acid in the context of different overall reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-A resolution. The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits. The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn2+-bound structure. Thus, absence of the metal ion does not perturb the structure of the active site. The structures of enolase, mandelate racemase, and MLE were superimposed. A comparison of metal ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that the position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (ii) that the positions of catalytic residues in the protein scaffold are conserved, whereas their identities and roles in catalysis vary.

Insights into the mechanism of catalysis by the P-C bond-cleaving enzyme phosphonoacetaldehyde hydrolase derived from gene sequence analysis and mutagenesis.

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and inorganic phosphate. In this study, the genes encoding phosphonatase in Bacillus cereus and in Salmonella typhimurium were cloned for high-level expression in Escherichia coli. The kinetic properties of the purified, recombinant phosphonatases were determined. The Schiff base mechanism known to operate in the B. cereus enzyme was verified for the S. typhimurium enzyme by phosphonoacetaldehyde-sodium borohydride-induced inactivation and by site-directed mutagenesis of the catalytic lysine 53. The protein sequence inferred from the B. cereus phosphonatase gene was determined, and this sequence was used along with that from the S. typhimurium phosphonatase gene sequence to search the primary sequence databases for possible structural homologues. We found that phosphonatase belongs to a novel family of hydrolases which appear to use a highly conserved active site aspartate residue in covalent catalysis. On the basis of this finding and the known stereochemical course of phosphonatase-catalyzed hydrolysis at phosphorus (retention), we propose a mechanism which involves Schiff base formation with lysine 53 followed by phosphoryl transfer to aspartate (at position 11 in the S. typhimurium enzyme and position 12 in the B. cereusphosphonatase) and last hydrolysis at the imine C(1) and acyl phosphate phosphorus.

Cloning and functional characterization of a rat renal organic cation transporter isoform (rOCT1A).

Polyspecific organic cation transporters in the renal proximal tubule mediate the secretion of many clinically used drugs as well as endogenous metabolites. Recently, two organic cation transporters (rOCT1 and rOCT2) were cloned from rat kidney. In this study, we report the cloning and functional expression of an rOCT1 isoform, rOCT1A, from rat kidney. Genomic DNA cloning and sequencing demonstrated that rOCT1A is an alternatively spliced variant of rOCT1 with a deletion of 104 base pairs near the 5'-end. The uptake of [14C]tetraethylammonium (TEA) in oocytes injected with the cRNA-encoding rOCT1A was increased 16-fold over that in water-injected oocytes (29 +/- 2.8 pmol/oocyte/h versus 1.8 +/- 0.13 pmol/oocyte/h, mean +/- S.E., p < 0.05). [14C]TEA uptake in the cRNA-injected oocytes was saturable (Km = 42 +/- 11 microM) and was inhibited significantly by organic cations, including cimetidine and N1-methylnicotinamide. The amino acid sequence was deduced from the cDNA after examination of all three reading frames. Two overlapping open reading frames were found. Studies with synthetic constructs suggest that a functional organic cation transporter is encoded by the larger open reading frame. The larger open reading frame encodes a 430-amino acid protein (termed rOCT1A) that is 92% identical to rOCT1 and 57% identical to rOCT2. From hydropathy analysis, rOCT1A is predicted to have 10 transmembrane domains with both amino and carboxyl termini intracellular. RNase protection assays demonstrate the presence of rOCT1A mRNA transcripts in rat kidney cortex, medulla, and intestine. These studies demonstrate the presence of a functional, alternatively spliced organic cation transporter (rOCT1A) in rat kidney.