RESUMEN
Clinical genetic variant classification science is a growing subspecialty of clinical genetics and genomics. The field's continued improvement is essential for the success of precision medicine in both germline (hereditary) and somatic (oncology) contexts. This review focuses on variant classification for DNA next-generation sequencing tests. We first summarize current limitations in variant discovery and definition, and then describe the current five- and four-tier classification systems outlined in dominant standards and guideline publications for germline and somatic tests, respectively. We then discuss measures of variant classification discordance and the field's bias for positive results, as well as considerations for panel size and population screening in the context of estimates of positive predictive value thatincorporate estimated variant classification imperfections. Finally, we share opinions on the current state of variant classification from some of the authors of the most widely used standards and guideline publications and from other domain experts.
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Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Genómica , Humanos , Medicina de PrecisiónRESUMEN
Missense variants represent a significant proportion of variants identified in clinical genetic testing. In the absence of strong clinical or functional evidence, the American College of Medical Genetics recommends that these findings be classified as variants of uncertain significance (VUS). VUSs may be reclassified to better inform patient care when new evidence is available. It is critical that the methods used for reclassification are robust in order to prevent inappropriate medical management strategies and unnecessary, life-altering surgeries. In an effort to provide evidence for classification, several in silico algorithms have been developed that attempt to predict the functional impact of missense variants through amino acid sequence conservation analysis. We report an analysis comparing internally derived, evidence-based classifications with the results obtained from six commonly used algorithms. We compiled a dataset of 1118 variants in BRCA1, BRCA2, MLH1, and MSH2 previously classified by our laboratory's evidence-based variant classification program. We compared internally derived classifications with those obtained from the following in silico tools: Align-GVGD, CONDEL, Grantham Analysis, MAPP-MMR, PolyPhen-2, and SIFT. Despite being based on similar underlying principles, all algorithms displayed marked divergence in accuracy, specificity, and sensitivity. Overall, accuracy ranged from 58.7 to 90.8% while the Matthews Correlation Coefficient ranged from 0.26-0.65. CONDEL, a weighted average of multiple algorithms, did not perform significantly better than its individual components evaluated here. These results suggest that the in silico algorithms evaluated here do not provide reliable evidence regarding the clinical significance of missense variants in genes associated with hereditary cancer.
RESUMEN
Mismatch repair prevents the accumulation of erroneous insertions/deletions and non-Watson-Crick base pairs in the genome. Pathogenic mutations in the MLH1 gene are associated with a predisposition to Lynch and Turcot's syndromes. Although genetic testing for these mutations is available, robust classification of variants requires strong clinical and functional support. Here, the first structure of the N-terminus of human MLH1, determined by X-ray crystallography, is described. The structure shares a high degree of similarity with previously determined prokaryotic MLH1 homologs; however, this structure affords a more accurate platform for the classification of MLH1 variants.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Nucleares/química , Proteínas Recombinantes de Fusión/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Clonación Molecular , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Homólogo 1 de la Proteína MutL , Proteínas Nucleares/genética , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Homología Estructural de ProteínaRESUMEN
Genetic variants of uncertain clinical significance (VUSs) are a common outcome of clinical genetic testing. Locus-specific variant databases (LSDBs) have been established for numerous disease-associated genes as a research tool for the interpretation of genetic sequence variants to facilitate variant interpretation via aggregated data. If LSDBs are to be used for clinical practice, consistent and transparent criteria regarding the deposition and interpretation of variants are vital, as variant classifications are often used to make important and irreversible clinical decisions. In this study, we performed a retrospective analysis of 2017 consecutive BRCA1 and BRCA2 genetic variants identified from 24,650 consecutive patient samples referred to our laboratory to establish an unbiased dataset representative of the types of variants seen in the US patient population, submitted by clinicians and researchers for BRCA1 and BRCA2 testing. We compared the clinical classifications of these variants among five publicly accessible BRCA1 and BRCA2 variant databases: BIC, ClinVar, HGMD (paid version), LOVD, and the UMD databases. Our results show substantial disparity of variant classifications among publicly accessible databases. Furthermore, it appears that discrepant classifications are not the result of a single outlier but widespread disagreement among databases. This study also shows that databases sometimes favor a clinical classification when current best practice guidelines (ACMG/AMP/CAP) would suggest an uncertain classification. Although LSDBs have been well established for research applications, our results suggest several challenges preclude their wider use in clinical practice.
RESUMEN
BACKGROUND: Cruzain, the major cysteine protease of Trypanosoma cruzi, is an essential enzyme for the parasite life cycle and has been validated as a viable target to treat Chagas' disease. As a proof-of-concept, K11777, a potent inhibitor of cruzain, was found to effectively eliminate T. cruzi infection and is currently a clinical candidate for treatment of Chagas' disease. METHODOLOGY/PRINCIPAL FINDINGS: WRR-483, an analog of K11777, was synthesized and evaluated as an inhibitor of cruzain and against T. cruzi proliferation in cell culture. This compound demonstrates good potency against cruzain with sensitivity to pH conditions and high efficacy in the cell culture assay. Furthermore, WRR-483 also eradicates parasite infection in a mouse model of acute Chagas' disease. To determine the atomic-level details of the inhibitor interacting with cruzain, a 1.5 A crystal structure of the protease in complex with WRR-483 was solved. The structure illustrates that WRR-483 binds covalently to the active site cysteine of the protease in a similar manner as other vinyl sulfone-based inhibitors. Details of the critical interactions within the specificity binding pocket are also reported. CONCLUSIONS: We demonstrate that WRR-483 is an effective cysteine protease inhibitor with trypanocidal activity in cell culture and animal model with comparable efficacy to K11777. Crystallographic evidence confirms that the mode of action is by targeting the active site of cruzain. Taken together, these results suggest that WRR-483 has potential to be developed as a treatment for Chagas' disease.
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Antiprotozoarios/administración & dosificación , Antiprotozoarios/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Inhibidores de Cisteína Proteinasa/administración & dosificación , Inhibidores de Cisteína Proteinasa/farmacología , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Sulfonas/administración & dosificación , Sulfonas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Antiprotozoarios/síntesis química , Antiprotozoarios/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/metabolismo , Dipéptidos/administración & dosificación , Dipéptidos/síntesis química , Dipéptidos/metabolismo , Dipéptidos/farmacología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C3H , Modelos Moleculares , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Pruebas de Sensibilidad Parasitaria , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Sulfonas/síntesis química , Sulfonas/metabolismo , Resultado del Tratamiento , Compuestos de Vinilo/administración & dosificación , Compuestos de Vinilo/síntesis química , Compuestos de Vinilo/metabolismo , Compuestos de Vinilo/farmacologíaRESUMEN
BACKGROUND: Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei. METHODS AND FINDINGS: We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002. CONCLUSIONS: The mature domain of our TbCat*CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat*CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain*K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.
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Cisteína Endopeptidasas/química , Trypanosoma brucei brucei/química , Sitios de Unión , Cristalografía por Rayos X/métodos , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Dipéptidos/química , Dipéptidos/metabolismo , Modelos Moleculares , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , Sulfonas/química , Sulfonas/metabolismo , Trypanosoma brucei brucei/enzimologíaRESUMEN
Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.
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Amebiasis/parasitología , Proteasas de Cisteína/química , Proteasas de Cisteína/fisiología , Entamoeba histolytica/metabolismo , Animales , Línea Celular Tumoral , Diseño de Fármacos , Regulación Enzimológica de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Cinética , Espectrometría de Masas/métodos , Ratones , Ratones Endogámicos C3H , Péptido Hidrolasas/química , Inhibidores de Proteasas/farmacología , Proteínas Recombinantes/química , Tiorredoxinas/químicaRESUMEN
A century after discovering that the Trypanosoma cruzi parasite is the etiological agent of Chagas disease, treatment is still plagued by limited efficacy, toxicity, and the emergence of drug resistance. The development of inhibitors of the major T. cruzi cysteine protease, cruzain, has been demonstrated to be a promising drug discovery avenue for this neglected disease. Here we establish that a nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitor substantially ameliorates symptoms of acute Chagas disease in a mouse model with no apparent toxicity. A high-resolution crystal structure confirmed the mode of inhibition and revealed key binding interactions of this novel inhibitor class. Subsequent structure-guided optimization then resulted in inhibitor analogues with improvements in potency despite minimal or no additions in molecular weight. Evaluation of the analogues in cell culture showed enhanced activity. These results suggest that nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors have the potential to fulfill the urgent need for improved Chagas disease chemotherapy.
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Enfermedad de Chagas/tratamiento farmacológico , Cetonas/síntesis química , Proteínas Protozoarias/antagonistas & inhibidores , Tripanocidas/síntesis química , Animales , Bovinos , Células Cultivadas , Cisteína Endopeptidasas , Femenino , Cetonas/química , Cetonas/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Ratones , Ratones Endogámicos C3H , Modelos Moleculares , Pruebas de Sensibilidad Parasitaria , Quinolinas/síntesis química , Quinolinas/química , Quinolinas/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/química , Triazoles/farmacología , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.
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Dihidropteroato Sintasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Pterinas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Modelos Moleculares , Estructura Molecular , Pterinas/química , Relación Estructura-ActividadRESUMEN
We describe here the identification of non-peptidic vinylsulfones that inhibit parasite cysteine proteases in vitro and inhibit the growth of Trypanosoma brucei brucei parasites in culture. A high resolution (1.75 A) co-crystal structure of 8a bound to cruzain reveals how the non-peptidic P2/P3 moiety in such analogs bind the S2 and S3 subsites of the protease, effectively recapitulating important binding interactions present in more traditional peptide-based protease inhibitors and natural substrates.
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Amidas/química , Proteasas de Cisteína/química , Inhibidores de Proteasas/química , Sulfonas/química , Tripanocidas/química , Amidas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Proteasas de Cisteína/metabolismo , Humanos , Células Jurkat , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/toxicidad , Estructura Terciaria de Proteína , Sulfonas/síntesis química , Sulfonas/farmacología , Sulfonas/toxicidad , Tripanocidas/síntesis química , Tripanocidas/toxicidad , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.
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Antiparasitarios/química , Cisteína Endopeptidasas/química , Plasmodium falciparum/enzimología , Inhibidores de Proteasas/química , Proteínas Protozoarias/química , Sulfonas/química , Trypanosoma brucei brucei/enzimología , Trypanosoma cruzi/enzimología , Animales , Antiparasitarios/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/enzimología , Cristalografía por Rayos X , Diseño de Fármacos , Cinética , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Inhibidores de Proteasas/uso terapéutico , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/antagonistas & inhibidores , Sulfonas/uso terapéutico , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/enzimologíaRESUMEN
The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1) - P(4) amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2) position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18)O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2) Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.
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Cisteína Endopeptidasas/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Animales , Antimaláricos/uso terapéutico , Cisteína Endopeptidasas/genética , Hemoglobinas/química , Humanos , Hidrólisis , Leucina/genética , Leucina/metabolismo , Malaria/tratamiento farmacológico , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/genética , Péptidos/metabolismo , Plasmodium falciparum/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Falcipain-2 and falcipain-3 are critical hemoglobinases of Plasmodium falciparum, the most virulent human malaria parasite. We have determined the 2.9 A crystal structure of falcipain-2 in complex with the epoxysuccinate E64 and the 2.5 A crystal structure of falcipain-3 in complex with the aldehyde leupeptin. These complexes represent the first crystal structures of plasmodial cysteine proteases with small molecule inhibitors and the first reported crystal structure of falcipain-3. Our structural analyses indicate that the relative shape and flexibility of the S2 pocket are affected by a number of discrete amino acid substitutions. The cumulative effect of subtle differences, including those at "gatekeeper" positions, may explain the observed kinetic differences between these two closely related enzymes.
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Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/química , Leupeptinas/química , Animales , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Plasmodium falciparum/enzimología , Especificidad por SustratoRESUMEN
The uvsWXY system is implicated in the replication and repair of the bacteriophage T4 genome. Whereas the roles of the recombinase (UvsX) and the recombination mediator protein (UvsY) are known, the precise role of UvsW is unclear. Sequence analysis identifies UvsW as a member of the monomeric SF2 helicase superfamily that translocates nucleic acid substrates via the action of two RecA-like motor domains. Functional homologies to Escherichia coli RecG and biochemical analyses have shown that UvsW interacts with branched nucleic acid substrates, suggesting roles in recombination and the rescue of stalled replication forks. A sequencing error at the 3'-end of the uvsW gene has revealed a second, short open reading frame that encodes a protein of unknown function called UvsW.1. We have determined the crystal structure of UvsW to 2.7A and the NMR solution structure of UvsW.1. UvsW has a four-domain architecture with structural homology to the eukaryotic SF2 helicase, Rad54. A model of the UvsW-ssDNA complex identifies structural elements and conserved residues that may interact with nucleic acid substrates. The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases.
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Bacteriófago T4/enzimología , ADN Helicasas/química , Proteínas Virales/química , Cristalografía por Rayos X , ADN Helicasas/metabolismo , Reparación del ADN/fisiología , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Células Eucariotas/enzimología , Genoma Viral/fisiología , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , RecQ Helicasas/química , RecQ Helicasas/metabolismo , Recombinación Genética/fisiología , Homología Estructural de Proteína , Proteínas Virales/metabolismo , Replicación Viral/fisiologíaRESUMEN
Information processing pathways such as DNA replication are conserved in eukaryotes and archaea and are significantly different from those found in bacteria. Single-stranded DNA-binding (SSB) proteins (or replication protein A, RPA, in eukaryotes) play a central role in many of these pathways. However, whilst euryarchaea have a eukaryotic-type RPA homologue, crenarchaeal SSB proteins appear much more similar to the bacterial proteins, with a single OB fold for DNA binding and a flexible C-terminal tail that is implicated in protein-protein interactions. We have determined the crystal structure of the SSB protein from the crenarchaeote Sulfolobus solfataricus to 1.26 A. The structure shows a striking and unexpected similarity to the DNA-binding domains of human RPA, providing confirmation of the close relationship between archaea and eukaryotes. The high resolution of the structure, together with thermodynamic and mutational studies of DNA binding, allow us to propose a molecular basis for DNA binding and define the features required for eukaryotic and archaeal OB folds.
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Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , ADN de Archaea/genética , ADN de Archaea/metabolismo , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicación A , Homología de Secuencia de Aminoácido , Sulfolobus/genética , Sulfolobus/metabolismo , TermodinámicaRESUMEN
dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) catalyzes the final step in the conversion of dTDP-D-glucose to dTDP-L-rhamnose in an NAD(P)H- and Mg2+-dependent reaction. L-rhamnose biosynthesis is an antibacterial target. The structure of RmlD from Salmonella enterica serovar Typhimurium has been determined, and complexes with NADH, NADPH, and dTDP-L-rhamnose are reported. RmlD differs from other short chain dehydrogenases in that it has a novel dimer interface that contains Mg2+. Enzyme catalysis involves hydride transfer from the nicotinamide ring of the cofactor to the C4'-carbonyl group of the substrate. The substrate is activated through protonation by a conserved tyrosine. NAD(P)H is bound in a solvent-exposed cleft, allowing facile replacement. We suggest a novel role for the conserved serine/threonine residue of the catalytic triad of SDR enzymes.