Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP

WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments.

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Expanding Substrate Specificity of ω-Transaminase by Rational Remodeling of a Large Substrate-Binding Pocket

Advanced Synthesis & Catalysis ◽

10.1002/adsc.201500239 ◽

2015 ◽

Vol 357 (12) ◽

pp. 2712-2720 ◽

Cited By ~ 20

Author(s):

Sang-Woo Han ◽

Eul-Soo Park ◽

Joo-Young Dong ◽

Jong-Shik Shin

Keyword(s):

Substrate Specificity ◽

Substrate Binding ◽

Binding Pocket ◽

Substrate Binding Pocket

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Evolutionarily Divergent Extradiol Dioxygenases Possess Higher Specificities for Polychlorinated Biphenyl Metabolites

Journal of Bacteriology ◽

10.1128/jb.187.2.415-421.2005 ◽

2005 ◽

Vol 187 (2) ◽

pp. 415-421 ◽

Cited By ~ 20

Author(s):

Pascal D. Fortin ◽

Andy T.-F. Lo ◽

María-Amparo Haro ◽

Stefan R. Kaschabek ◽

Walter Reineke ◽

...

Keyword(s):

Polychlorinated Biphenyl ◽

Substrate Binding ◽

Structural Data ◽

Binding Pocket ◽

Distal Portion ◽

The Other ◽

Substrate Binding Pocket ◽

Dihydroxybiphenyl Dioxygenase ◽

Extradiol Dioxygenases ◽

Better Than

ABSTRACT The reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di-, and trichlorinated (triCl) 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia sp. strain LB400 (DHBDLB400), DHBDP6-I and DHBDP6-III from Rhodococcus globerulus P6, and 2,2′,3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. strain RW1 (THBDRW1). The specificity of each isozyme for particular DHBs differed by up to 3 orders of magnitude. Interestingly, the K m app values of each isozyme for the tested polychlorinated DHBs were invariably lower than those of monochlorinated DHBs. Moreover, each enzyme cleaved at least one of the tested chlorinated (Cl) DHBs better than it cleaved DHB (e.g., apparent specificity constants for 3′,5′-dichlorinated [diCl] DHB were 2 to 13.4 times higher than for DHB). These results are consistent with structural data and modeling studies which indicate that the substrate-binding pocket of the DHBDs is hydrophobic and can accommodate the Cl DHBs, particularly in the distal portion of the pocket. Although the activity of DHBDP6-III was generally lower than that of the other three enzymes, six of eight tested Cl DHBs were better substrates for DHBDP6-III than was DHB. Indeed, DHBDP6-III had the highest apparent specificity for 4,3′,5′-triCl DHB and cleaved this compound better than two of the other enzymes. Of the four enzymes, THBDRW1 had the highest specificity for 2′-Cl DHB and was at least five times more resistant to inactivation by 2′-Cl DHB, consistent with the similarity between the latter and 2,2′,3-trihydroxybiphenyl. Nonetheless, THBDRW1 had the lowest specificity for 2′,6′-diCl DHB and, like the other enzymes, was unable to cleave this critical PCB metabolite (k cat app < 0.001 s−1).

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Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2113573118 ◽

2021 ◽

Vol 118 (49) ◽

pp. e2113573118

Author(s):

Carlos F. Rodriguez ◽

Paloma Escudero-Bravo ◽

Lucía Díaz ◽

Paola Bartoccioni ◽

Carmen García-Martín ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Substrate Specificity ◽

Molecular Mechanisms ◽

Substrate Binding ◽

Binding Pocket ◽

Neutral Amino Acid ◽

Substrate Preference ◽

Structural Basis ◽

Substrate Binding Pocket

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

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Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-β-Lactamases

Frontiers in Microbiology ◽

10.3389/fmicb.2021.752535 ◽

2022 ◽

Vol 12 ◽

Author(s):

Yeongjin Yun ◽

Sangjun Han ◽

Yoon Sik Park ◽

Hyunjae Park ◽

Dogyeong Kim ◽

...

Keyword(s):

Substrate Specificity ◽

Active Site ◽

Chemical Structure ◽

Substrate Binding ◽

Binding Pocket ◽

Structural Features ◽

Amino Acid Sequences ◽

Zinc Ions ◽

Substrate Binding Pocket ◽

Inhibitory Spectrum

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.

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Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

Nature Communications ◽

10.1038/s41467-020-20675-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yufei Han ◽

Qian Zhuang ◽

Bo Sun ◽

Wenping Lv ◽

Sheng Wang ◽

...

Keyword(s):

Crystal Structure ◽

Biochemical Characterization ◽

Substrate Binding ◽

Binding Pocket ◽

Substrate Binding Pocket ◽

Transmembrane Segments ◽

Therapeutic Molecules ◽

Binding Cavity ◽

Immune System Regulation ◽

Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

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