substrate specificity
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2022 ◽  
Vol 12 ◽  
Author(s):  
Yeongjin Yun ◽  
Sangjun Han ◽  
Yoon Sik Park ◽  
Hyunjae Park ◽  
Dogyeong Kim ◽  
...  

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central β-sheets in the conserved αβ/βα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of β-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of β-lactam-bound MBLs show that the binding of β-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides β-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.


2022 ◽  
Author(s):  
Jai Krishna Mahto ◽  
Neetu Neetu ◽  
Monica Sharma ◽  
Monika Dubey ◽  
Bhanu Prakash Vellanki ◽  
...  

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.


2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Ahjin Jang ◽  
Dasom Cheon ◽  
Eunha Hwang ◽  
Yangmee Kim

AbstractTo survive in diverse environments, bacteria adapt by changing the composition of their cell membrane fatty acids. Compared with aerobic bacteria, Cutibacterium acnes has much greater contents of branched-chain fatty acids (BCFAs) in the cell membrane, which helps it survive in anaerobic environments. To synthesize BCFAs, C. acnes acyl carrier protein (CaACP) has to transfer growing branched acyl intermediates from its hydrophobic cavity to fatty acid synthases. CaACP contains an unconserved, distinctive Cys50 in its hydrophobic pocket, which corresponds to Leu in other bacterial acyl carrier proteins (ACPs). Herein, we investigated the substrate specificity of CaACP and the importance of Cys50 in its structural stability. We mutated Cys50 to Leu (C50L mutant) and measured the melting temperatures (Tms) of both CaACP and the C50L mutant by performing circular dichroism experiments. The Tm of CaACP was very low (49.6 °C), whereas that of C50L mutant was 55.5 °C. Hydrogen/deuterium exchange experiments revealed that wild-type CaACP showed extremely fast exchange rates within 50 min, whereas amide peaks of the C50L mutant in the heteronuclear single quantum coherence spectrum remained up to 200 min, thereby implying that Cys50 is the key residue contributing to the structural stability of CaACP. We also monitored chemical shift perturbations upon apo to holo, apo to butyryl, and apo to isobutyryl conversion, confirming that CaACP can accommodate isobutyryl BCFAs. These results provide a preliminary understanding into the substrate specificity of CaACPs for the production of BCFAs necessary to maintain cell membrane fluidity under anaerobic environments.


2022 ◽  
Vol 3 (1) ◽  
pp. 11-23
Author(s):  
Fabrice Tsigaing Tsigain ◽  
Blondo-Pascal Metsebing ◽  
Dominique Claude Mossebo ◽  
Leif Randulff Ryvarden ◽  
Romuald Oba ◽  
...  

A total of 72 species of wood-rotting Basidiomycetes belonging to 40 genera, 13 families and 5 orders, were investigated in this study of which 46 for the first time as far as type of wood-rot is concerned. 61 of the 72 cause white rot (W) representing 84.72 % of the total and 11 cause brown rot (B) or just 15.27 %, confirming the predominance already known of white rot (W) on brown rot (B) as shown in other studies. Results recorded show that even though most species belonging to same genera display the same type of rot (W or B), species of some few other genera were found to rather display different types of rot (W and B) in species within the same genera. Other results show that when determining the type of wood-rot caused by some species of fungi, in case of negative (-) test using tincture of guaiac which is known as the key test to determine the type of rot, syringaldazine must also be used as a differential test before concluding whether the species is a white (W) or brown rot (B) fungus. The level of activity of tyrosinase and peroxidase shows important variations between species of some genera, whereby some species of a given genus show varying potential level (+, ++, +++, ++++) of activities of these two enzymes, whereas other species of the same genus show no sign (-) of activity of the same enzymes. Therefore, our results led to the conclusion that the presence (+, ++, +++, ++++) or absence (-) of activity of peroxidase and tyrosinase can from now onwards also be used as an enzyme linked taxonomic criteria to distinguish between species of some genera. About detection of laccases activity, if a negative (-) result occurs during detection test in a wood-rotting fungus using syringaldazine, α-naphtol must also be used as a differential test before concluding on whether the species produces laccases or not. Based on the overall results recorded in the detection of enzymes activities, it appears necessary to use, where indicated, several substrates with different chemical sensitivities to detect the existence of an enzyme and its potential activity level in a fungal species. Additionally, preliminary lists of wood-decay fungi with potentially strong (+++, ++++) capacity to produce different types of polyphenol oxidases potentially usable in paper making industries, wastewater treatment and soil remediation, are provided. With regard to the study of substrate specificity which aimed at finding out the existence or not of a specificity between polyporales and tree wood species on which they grow, the first results recorded on a limited number of trees investigated led to the preliminary conclusion that, although some few tree species may serve as hosts for only a single species of polyporales, there is rather a greater tendency of finding several species growing on different species of wood as well as the same species of wood hosting several species of polyporales. These field observations led to the remark that a much larger inventory including a greater number of tree species in various tropical ecosystems is necessary in order to come out with a final conclusion.


Author(s):  
Jaysón Davidson ◽  
Kyndall Nicholas ◽  
Jeremy Young ◽  
Deborah G. Conrady ◽  
Stephen Mayclin ◽  
...  

Paraburkholderia xenovorans degrades organic wastes, including polychlorinated biphenyls. The atomic structure of a putative dehydrogenase/reductase (SDR) from P. xenovorans (PxSDR) was determined in space group P21 at a resolution of 1.45 Å. PxSDR shares less than 37% sequence identity with any known structure and assembles as a prototypical SDR tetramer. As expected, there is some conformational flexibility and difference in the substrate-binding cavity, which explains the substrate specificity. Uniquely, the cofactor-binding cavity of PxSDR is not well conserved and differs from those of other SDRs. PxSDR has an additional seven amino acids that form an additional unique loop within the cofactor-binding cavity. Further studies are required to determine how these differences affect the enzymatic functions of the SDR.


2022 ◽  
pp. 101585
Author(s):  
Yuko Ohara Nemoto ◽  
Yu Shimoyama ◽  
Toshio Ono ◽  
Mohammad Tanvir Sarwar ◽  
Manami Nakasato ◽  
...  

Life ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 32
Author(s):  
Kohtoh Yukawa ◽  
Ryo Mizuuchi ◽  
Norikazu Ichihashi

A change from RNA- to DNA-based genetic systems is hypothesized as a major transition in the evolution of early life forms. One of the possible requirements for this transition is a change in the substrate specificity of the replication enzyme. It is largely unknown how such changes would have occurred during early evolutionary history. In this study, we present evidence that an RNA replication enzyme that has evolved in the absence of deoxyribonucleotide triphosphates (dNTPs) relaxes its substrate specificity and incorporates labeled dNTPs. This result implies that ancient replication enzymes, which probably evolved in the absence of dNTPs, could have incorporated dNTPs to synthesize DNA soon after dNTPs became available. The transition from RNA to DNA, therefore, might have been easier than previously thought.


Holzforschung ◽  
2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Dharshana Padmakshan ◽  
Vitaliy I. Timokhin ◽  
Fachuang Lu ◽  
Paul F. Schatz ◽  
Ruben Vanholme ◽  
...  

Abstract Hydroxycinnamoyl shikimates were reported in 2005 to be intermediates in monolignol biosynthesis. 3-Hydroxylation of p-coumarate, originally thought to occur via coumarate 3-hydroxylase (C3H) from p-coumaric acid or its CoA thioester, was revealed to be via the action of coumaroyl shikimate 3′-hydroxylase (C3′H) utilizing p-coumaroyl shikimate as the substrate, itself derived from p-coumaroyl-CoA via hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase (HCT). The same HCT was conjectured to convert the product, caffeoyl shikimate, to caffeoyl-CoA to continue on the pathway starting with its 3-O-methylation. At least in some plants, however, a more recently discovered caffeoyl shikimate esterase (CSE) enzyme hydrolyzes caffeoyl shikimate to caffeic acid from which it must again produce its CoA thioester to continue on the monolignol biosynthetic pathway. HCT and CSE are therefore monolignol biosynthetic pathway enzymes that have provided new opportunities to misregulate lignification. To facilitate studies into the action and substrate specificity of C3H/C3′H, HCT, and CSE enzymes, as well as for metabolite authentication and for enzyme characterization, including kinetics, a source of authentic substrates and products was required. A synthetic scheme starting from commercially available shikimic acid and the four key hydroxycinnamic acids (p-coumaric, caffeic, ferulic, and sinapic acid) has been developed to provide this set of hydroxycinnamoyl shikimates for researchers.


2021 ◽  
pp. 26-32
Author(s):  
Qiangwei Wang ◽  
Qingshi Wu ◽  
Ting Ye ◽  
Xiaofei Wang ◽  
Huijuan Qiu ◽  
...  

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