scholarly journals Cloning of a Novel Nicotine Oxidase Gene from Pseudomonas sp. Strain HZN6 Whose Product Nonenantioselectively Degrades Nicotine to Pseudooxynicotine

2013 ◽  
Vol 79 (7) ◽  
pp. 2164-2171 ◽  
Author(s):  
Jiguo Qiu ◽  
Yun Ma ◽  
Jing Zhang ◽  
Yuezhong Wen ◽  
Weiping Liu
Keyword(s):  
Amine Oxidase ◽  
Sole Source ◽  
Site Directed Mutagenesis ◽  
Broad Host Range ◽  
Upstream Sequence ◽  
Oxidase Gene ◽  
Content Type ◽  
Reading Frame ◽  
Degrading Bacteria ◽  
Nicotine Degradation

ABSTRACTPseudomonassp. strain HZN6 utilizes nicotine as its sole source of carbon, nitrogen, and energy. However, its catabolic mechanism has not been elucidated. In this study, self-formed adaptor PCR was performed to amplify the upstream sequence of the pseudooxynicotine amine oxidase gene. A 1,437-bp open reading frame (designatednox) was found to encode a nicotine oxidase (NOX) that shows 30% amino acid sequence identity with 6-hydroxy-l-nicotine oxidase fromArthrobacter nicotinovorans. Thenoxgene was cloned into a broad-host-range cloning vector and transferred into the non-nicotine-degrading bacteriaEscherichia coliDH5α (DH-nox) andPseudomonas putidaKT2440 (KT-nox). The transconjugant KT-nox obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine, while DH-nox did not. Reverse transcription-PCR showed that thenoxgene is expressed in both DH5α and KT2440, suggesting that additional factors required for nicotine degradation are present in aPseudomonasstrain(s), but not inE. coli. The mutant of strain HZN6 withnoxdisrupted lost the ability to degrade nicotine, but not pseudooxynicotine. These results suggested that thenoxgene is responsible for the first step of nicotine degradation. The (RS)-nicotine degradation results showed that the two enantiomers were degraded at approximately the same rate, indicating that NOX does not show chiral selectivity. Site-directed mutagenesis revealed that both the conserved flavin adenine dinucleotide (FAD)-binding GXGXXG motif and His456 are essential for nicotine degradation activity.

scholarly journals Molecular Mechanism of Nicotine Degradation by a Newly Isolated Strain, Ochrobactrum sp. Strain SJY1

2014 ◽  
Vol 81 (1) ◽  
pp. 272-281 ◽  
Author(s):  
Hao Yu ◽  
Hongzhi Tang ◽  
Xiongyu Zhu ◽  
Yangyang Li ◽  
Ping Xu
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequence Identity ◽  
Specific Activity ◽  
Amine Oxidase ◽  
Sole Source ◽  
Content Type ◽  
Sequence Identity ◽  
Nicotine Degradation ◽  
Functional Pathway

ABSTRACTA newly isolated strain, SJY1, identified asOchrobactrumsp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designatedvppB,vppD, andvppE, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. ThevppBgene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-N-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-l-nicotine oxidase, 24%) from the pyridine pathway. ThevppDgene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) fromPseudomonas putidastrain S16, whereas the specific activity of VppD is ∼10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation.

scholarly journals Structural Insights into 6-Hydroxypseudooxynicotine Amine Oxidase from Pseudomonas geniculata N1, the Key Enzyme Involved in Nicotine Degradation

2020 ◽  
Vol 86 (19) ◽  
Author(s):  
Gongquan Liu ◽  
Weiwei Wang ◽  
Fangyuan He ◽  
Peng Zhang ◽  
Ping Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Information ◽  
Amine Oxidase ◽  
Substrate Binding ◽  
Docking Study ◽  
Site Directed Mutagenesis ◽  
Flavin Mononucleotide ◽  
Structure Comparison ◽  
Content Type ◽  
Key Enzyme

ABSTRACT Bacteria degrade nicotine mainly using pyridine and pyrrolidine pathways. Previously, we discovered a hybrid of the pyridine and pyrrolidine pathways (the VPP pathway) in Pseudomonas geniculata N1 and characterized its key enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD). It catalyzes oxidative deamination of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde-pyridine, which is the crucial step connecting upstream and downstream portions of the VPP pathway. We determined the crystal structure of wild-type HisD to 2.6 Å. HisD is a monomer that contains a flavin mononucleotide, an iron-sulfur cluster, and ADP. On the basis of sequence alignment and structure comparison, a difference has been found among HisD, closely related trimethylamine dehydrogenase (TMADH), and histamine dehydrogenase (HADH). The flavin mononucleotide (FMN) cofactor is not covalently bound to any residue, and the FMN isoalloxazine ring is planar in HisD compared to TMADH or HADH, which forms a 6-S-cysteinyl flavin mononucleotide cofactor and has an FMN isoalloxazine ring in a “butterfly bend” conformation. Based on the structure, docking study, and site-directed mutagenesis, the residues Glu60, Tyr170, Asp262, and Trp263 may be involved in substrate binding. The expanded understanding of the substrate binding mode from this study may guide rational engineering of such enzymes for biodegradation of potential pollutants or for bioconversion to generate desired products. IMPORTANCE Nicotine is a major tobacco alkaloid in tobacco waste. Pyridine and pyrrolidine pathways are the two best-elucidated nicotine metabolic pathways; Pseudomonas geniculata N1 catabolizes nicotine via a hybrid between the pyridine and pyrrolidine pathways. The crucial enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD), links the upstream and downstream portions of the VPP pathway; however, there is little structural information about this important enzyme. In this study, we determined the crystal structure of HisD from Pseudomonas geniculata N1. Its basic insights about the structure may help us to guide the engineering of such enzymes for bioremediation and bioconversion applications.

scholarly journals Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

2015 ◽  
Vol 81 (24) ◽  
pp. 8330-8338 ◽  
Author(s):  
Hao Yu ◽  
Hongzhi Tang ◽  
Yangyang Li ◽  
Ping Xu
Keyword(s):  
Inductively Coupled Plasma ◽  
Large Subunit ◽  
Sole Source ◽  
Small Subunit ◽  
Binding Domain ◽  
Mass Spectrometry Analysis ◽  
Resting Cells ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Nicotine Degradation

ABSTRACTOchrobactrumsp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppASandvppAL[subunits S and L, respectively]). ThevppAgenes were heterologously expressed in the non-nicotine-degrading strainsEscherichia coliDH5α andPseudomonas putidaKT2440; only thePseudomonasstrain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.

Effects of Bacterial Host and Dichloromethane Dehalogenase on the Competitiveness of Methylotrophic Bacteria Growing with Dichloromethane

1998 ◽  
Vol 64 (4) ◽  
pp. 1194-1202 ◽  
Author(s):  
Daniel Gisi ◽  
Laurent Willi ◽  
Hubert Traber ◽  
Thomas Leisinger ◽  
Stéphane Vuilleumier
Keyword(s):  
Growth Rates ◽  
Sewage Treatment ◽  
Selective Advantage ◽  
Sole Source ◽  
Kinetic Properties ◽  
Broad Host Range ◽  
Methylotrophic Bacteria ◽  
Maximal Growth ◽  
Broad Host Range Plasmid ◽  
Degrading Bacteria

ABSTRACT Methylobacterium sp. strain DM4 andMethylophilus sp. strain DM11 can grow with dichloromethane (DCM) as the sole source of carbon and energy by virtue of homologous glutathione-dependent DCM dehalogenases with markedly different kinetic properties (the k cat values of the enzymes of these strains are 0.6 and 3.3 s−1, respectively, and theKm values are 9 and 59 μM, respectively). These strains, as well as transconjugant bacteria expressing the DCM dehalogenase gene (dcmA) from DM11 or DM4 on a broad-host-range plasmid in the background of dcmA mutant DM4-2cr, were investigated by growing them under growth-limiting conditions and in the presence of an excess of DCM. The maximal growth rates and maximal levels of dehalogenase for chemostat-adapted bacteria were higher than the maximal growth rates and maximal levels of dehalogenase for batch-grown bacteria. The substrate saturation constant of strain DM4 was much lower than theKm of its associated dehalogenase, suggesting that this strain is adapted to scavenge low concentrations of DCM. Strains and transconjugants expressing the DCM dehalogenase from strain DM11, on the other hand, had higher growth rates than bacteria expressing the homologous dehalogenase from strain DM4. Competition experiments performed with pairs of DCM-degrading strains revealed that a strain expressing the dehalogenase from DM4 had a selective advantage in continuous culture under substrate-limiting conditions, while strains expressing the DM11 dehalogenase were superior in batch culture when there was an excess of substrate. Only DCM-degrading bacteria with a dcmA gene similar to that from strain DM4, however, were obtained in batch enrichment cultures prepared with activated sludge from sewage treatment plants.

scholarly journals Microbial Community Degradation of Widely Used Quaternary Ammonium Disinfectants

2014 ◽  
Vol 80 (19) ◽  
pp. 5892-5900 ◽  
Author(s):  
Seungdae Oh ◽  
Zohre Kurt ◽  
Despina Tsementzi ◽  
Michael R. Weigand ◽  
Minjae Kim ◽  
...  
Keyword(s):  
Microbial Community ◽  
Amine Oxidase ◽  
Aerobic Biodegradation ◽  
Genetic Determinants ◽  
Community Members ◽  
Oxidase Gene ◽  
Content Type ◽  
Biochemical Assays ◽  
Microbial Infections ◽  
By Products

ABSTRACTBenzalkonium chlorides (BACs) are disinfectants widely used in a variety of clinical and environmental settings to prevent microbial infections, and they are frequently detected in nontarget environments, such as aquatic and engineered biological systems, even at toxic levels. Therefore, microbial degradation of BACs has important ramifications for alleviating disinfectant toxicity in nontarget environments as well as compromising disinfectant efficacy in target environments. However, how natural microbial communities respond to BAC exposure and what genes underlie BAC biodegradation remain elusive. Our previous metagenomic analysis of a river sediment microbial community revealed that BAC exposure selected for a low-diversity community, dominated by several members of thePseudomonasgenus that quickly degraded BACs. To elucidate the genetic determinants of BAC degradation, we conducted time-series metatranscriptomic analysis of this microbial community during a complete feeding cycle with BACs as the sole carbon and energy source under aerobic conditions. Metatranscriptomic profiles revealed a candidate gene for BAC dealkylation, the first step in BAC biodegradation that results in a product 500 times less toxic. Subsequent biochemical assays and isolate characterization verified that the putative amine oxidase gene product was functionally capable of initiating BAC degradation. Our analysis also revealed cooperative interactions among community members to alleviate BAC toxicity, such as the further degradation of BAC dealkylation by-products by organisms not encoding amine oxidase. Collectively, our results advance the understanding of BAC aerobic biodegradation and provide genetic biomarkers to assess the critical first step of this process in nontarget environments.

scholarly journals A Novel (S)-6-Hydroxynicotine Oxidase Gene from Shinella sp. Strain HZN7

2014 ◽  
Vol 80 (18) ◽  
pp. 5552-5560 ◽  
Author(s):  
Jiguo Qiu ◽  
Yin Wei ◽  
Yun Ma ◽  
Rongti Wen ◽  
Yuezhong Wen ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Flavin Adenine Dinucleotide ◽  
Enzyme Level ◽  
Oxidase Gene ◽  
Content Type ◽  
Nicotine Degradation ◽  
Degradation Activity ◽  
Mutant Genes ◽  
Tobacco Waste

ABSTRACTNicotine is an important environmental toxicant in tobacco waste.Shinellasp. strain HZN7 can metabolize nicotine into nontoxic compounds via variations of the pyridine and pyrrolidine pathways. However, the catabolic mechanism of this variant pathway at the gene or enzyme level is still unknown. In this study, two 6-hydroxynicotine degradation-deficient mutants, N7-M9 and N7-W3, were generated by transposon mutagenesis. The corresponding mutant genes, designatednctBandtnp2, were cloned and analyzed. ThenctBgene encodes a novel flavin adenine dinucleotide-containing (S)-6-hydroxynicotine oxidase that converts (S)-6-hydroxynicotine into 6-hydroxy-N-methylmyosmine and then spontaneously hydrolyzes into 6-hydroxypseudooxynicotine. The deletion and complementation of thenctBgene showed that this enzyme is essential for nicotine or (S)-6-hydroxynicotine degradation. Purified NctB could also convert (S)-nicotine intoN-methylmyosmine, which spontaneously hydrolyzed into pseudooxynicotine. The kinetic constants of NctB toward (S)-6-hydroxynicotine (Km= 0.019 mM,kcat= 7.3 s−1) and nicotine (Km= 2.03 mM,kcat= 0.396 s−1) indicated that (S)-6-hydroxynicotine is the preferred substratein vivo. NctB showed no activities toward theRenantiomer of nicotine or 6-hydroxynicotine. Strain HZN7 could degrade (R)-nicotine into (R)-6-hydroxynicotine without any further degradation. Thetnp2gene from mutant N7-W3 encodes a putative transposase, and its deletion did not abolish the nicotine degradation activity. This study advances the understanding of the microbial diversity of nicotine biodegradation.

scholarly journals MdoR Is a Novel Positive Transcriptional Regulator for the Oxidation of Methanol in Mycobacterium sp. Strain JC1

2011 ◽  
Vol 193 (22) ◽  
pp. 6288-6294 ◽  
Author(s):  
Hyuk Park ◽  
Young T. Ro ◽  
Young M. Kim
Keyword(s):  
Methanol Oxidation ◽  
Promoter Region ◽  
Transcriptional Regulator ◽  
Sole Source ◽  
Binding Motif ◽  
Mobility Shift ◽  
Content Type ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Oxidation Of Methanol

Mycobacteriumsp. strain JC1 is able to grow on methanol as a sole source of carbon and energy using methanol:N,N′-dimethyl-4-nitrosoaniline oxidoreductase (MDO) as a key enzyme for methanol oxidation. The second open reading frame (mdoR) upstream of, and running divergently from, themdogene was identified as a gene for a TetR family transcriptional regulator. The N-terminal region of MdoR contained a helix-turn-helix DNA-binding motif. An electrophoretic mobility shift assay (EMSA) indicated that MdoR could bind to amdopromoter region containing an inverted repeat. ThemdoRdeletion mutant did not grow on methanol, but growth on methanol was restored by a plasmid containing an intactmdoRgene. In DNase I footprinting and EMSA experiments, MdoR bound to two inverted repeats in the putativemdoRpromoter region. Reverse transcription-PCR indicated that themdoRgene was transcribed only in cells growing on methanol, whereas β-galactosidase assays showed that themdoRpromoter was activated in the presence of methanol. These results indicate that MdoR serves as a transcriptional activator for the expression ofmdoand its own gene. Also, MdoR is the first discovered member of the TetR family of transcriptional regulators to be involved in the regulation of the methanol oxidation, as well as to function as a positive autoregulator.

scholarly journals The Structure of the Cysteine-Rich Domain of Plasmodium falciparum P113 Identifies the Location of the RH5 Binding Site

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Ivan Campeotto ◽  
Francis Galaway ◽  
Shahid Mehmood ◽  
Lea K. Barfod ◽  
Doris Quinkert ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Binding Site ◽  
Structural Information ◽  
Blood Stage ◽  
Site Directed Mutagenesis ◽  
Effective Vaccine ◽  
Binding Region ◽  
Fab Fragment ◽  
Content Type ◽  
Blood Stage Malaria

ABSTRACT Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5. IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum. It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it—and the other parasite proteins it interacts with—promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

scholarly journals Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Peter Mellroth ◽  
Tatyana Sandalova ◽  
Alexey Kikhney ◽  
Francisco Vilaplana ◽  
Dusan Hesek ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Solution Structure ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Lytic Activity ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Content Type ◽  
Peptidoglycan Synthesis

ABSTRACT The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

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