Regulation of active site coupling in glutamine-dependent NAD+ synthetase

LaRonde-LeBlanc, N.; Resto, Melissa; Gerratana, Barbara

doi:10.1038/nsmb.1567

Cited by 44 publications

(100 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The amino acid sequence of abNadE did not show appreciable similarity with the NMN synthetase subfamily but was rather characteristic of NAD synthetase with the additional glutamine transferase domain responsible for the utilization of glutamine as in eukaryotes (34) and some bacteria (e.g. Mycobacterium tuberculosis) (35). Nevertheless, this possibility could not be excluded without additional experimental analysis (see below).…”

Section: Resultsmentioning

confidence: 91%

Genomics-driven Reconstruction of Acinetobacter NAD Metabolism

Sorci

Blaby

Ingeniis

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Enzymes involved in the last steps of NAD biogenesis, nicotinate mononucleotide adenylyltransferase (NadD) and NAD synthetase (NadE), are conserved and essential in most bacterial species and are established targets for antibacterial drug development. Our genomics-based reconstruction of NAD metabolism in the emerging pathogen Acinetobacter baumannii revealed unique features suggesting an alternative targeting strategy. Indeed, genomes of all analyzed Acinetobacter species do not encode NadD, which is functionally replaced by its distant homolog NadM. We combined bioinformatics with genetic and biochemical techniques to elucidate this and other important features of Acinetobacter NAD metabolism using a model (nonpathogenic) strain Acinetobacter baylyi sp. ADP1. Thus, a comparative kinetic characterization of PncA, PncB, and NadV enzymes allowed us to suggest distinct physiological roles for the two alternative, deamidating and nondeamidating, routes of nicotinamide salvage/recycling. The role of the NiaP transporter in both nicotinate and nicotinamide salvage was confirmed. The nondeamidating route was shown to be transcriptionally regulated by an ADP-ribose-responsive repressor NrtR. The NadM enzyme was shown to possess dual substrate specificity toward both nicotinate and nicotinamide mononucleotide substrates, which is consistent with its essential role in all three routes of NAD biogenesis, de novo synthesis as well as the two salvage pathways. The experimentally confirmed unconditional essentiality of nadM provided support for the choice of the respective enzyme as a drug target. In contrast, nadE, encoding a glutamine-dependent NAD synthetase, proved to be dispensable when the nondeamidating salvage pathway functioned as the only route of NAD biogenesis.Acinetobacter baumannii is an emerging pathogen that belongs to a relatively underexplored branch of ␥-proteobacteria. It can cause severe pneumonia and infections of the urinary tract, bloodstream, and other parts of the body. Some isolates of A. baumannii display resistance to many known antibiotics (1, 2), emphasizing the importance of pursuing new therapeutic targets for drug development. Biogenesis of nicotinamide adenine nucleotide (NAD), an indispensable cofactor involved in a multitude of biochemical transformations in metabolic networks of all species, was recently established as a target pathway for the development of new antibiotics (3-7). Beyond its main function as a redox cofactor, NAD is consumed as a co-substrate by a number of nonredox enzymes such as bacterial DNA ligase and protein deacetylase of the CobB/Sir2 family (8, 9). A degradative consumption of NAD by these and, likely, other (not fully elucidated) enzymes demands continuous replenishing of the NAD pool, providing further rationale for targeting essential enzymes involved in its biogenesis and recycling. Among these enzymes, nicotinate mononucleotide adenylyltransferase (NaMNAT) 4 of the NadD family and NAD synthetase of the NadE family are widely recognized as the most promising...

show abstract

Section: Resultsmentioning

confidence: 91%

Genomics-driven Reconstruction of Acinetobacter NAD Metabolism

Sorci

Blaby

Ingeniis

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Thus, the ion pair could potentially act as a gate that opens and closes in response to product release and substrate binding, respectively. We note that there are several examples of gates and constrictions blocking channeling pathways in other bifunctional enzymes (33)(34)(35)(36).…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

Srivastava

Schuermann

White

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

130

View full text Add to dashboard Cite

The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD þ -dependent Δ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 Å resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 Å. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 Å and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Δ 1 -pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Δ 1 -pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.proline catabolism | substrate channeling

show abstract

“…Gating mechanisms are not unusual for channeling enzymes. For example, structural and kinetic analysis of glutamine-dependent NAD ϩ synthetase identified a gating role for a Tyr residue in the glutaminase active site (48,49). Tyr-58 is proposed to move and thereby allow ammonia to enter the 40 Å channeling pathway to the NAD ϩ synthetase active site (48).…”

mentioning

confidence: 99%

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

Moxley

Sanyal

Krishnan

et al. 2014

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background:PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism. Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step. Conclusion: Substrate channeling in PutA exhibits hysteretic behavior. Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation.

show abstract

Regulation of active site coupling in glutamine-dependent NAD+ synthetase

Cited by 44 publications

References 41 publications

Genomics-driven Reconstruction of Acinetobacter NAD Metabolism

Genomics-driven Reconstruction of Acinetobacter NAD Metabolism

Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

Contact Info

Product

Resources

About