Amino acid 310 determines the donor substrate specificity of serogroup W-135 and Y capsule polymerases ofNeisseria meningitidis

Abstract The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by co-expression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and non-substrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

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S-Acylation of Proteins of Coronavirus and Influenza Virus: Conservation of Acylation Sites in Animal Viruses and DHHC Acyltransferases in Their Animal Reservoirs

Pathogens ◽

10.3390/pathogens10060669 ◽

2021 ◽

Vol 10 (6) ◽

pp. 669

Author(s):

Dina A. Abdulrahman ◽

Xiaorong Meng ◽

Michael Veit

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Ion Channels ◽

Substrate Specificity ◽

Influenza A ◽

3D Structure ◽

Viral Fitness ◽

Amino Acid Substitutions ◽

Essential Function ◽

Animal Reservoirs

Recent pandemics of zoonotic origin were caused by members of coronavirus (CoV) and influenza A (Flu A) viruses. Their glycoproteins (S in CoV, HA in Flu A) and ion channels (E in CoV, M2 in Flu A) are S-acylated. We show that viruses of all genera and from all hosts contain clusters of acylated cysteines in HA, S and E, consistent with the essential function of the modification. In contrast, some Flu viruses lost the acylated cysteine in M2 during evolution, suggesting that it does not affect viral fitness. Members of the DHHC family catalyze palmitoylation. Twenty-three DHHCs exist in humans, but the number varies between vertebrates. SARS-CoV-2 and Flu A proteins are acylated by an overlapping set of DHHCs in human cells. We show that these DHHC genes also exist in other virus hosts. Localization of amino acid substitutions in the 3D structure of DHHCs provided no evidence that their activity or substrate specificity is disturbed. We speculate that newly emerged CoVs or Flu viruses also depend on S-acylation for replication and will use the human DHHCs for that purpose. This feature makes these DHHCs attractive targets for pan-antiviral drugs.

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LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity

Biochemical Journal ◽

10.1042/bj20060379 ◽

2006 ◽

Vol 398 (3) ◽

pp. 531-538 ◽

Cited By ~ 116

Author(s):

Yukiko Mizutani ◽

Akio Kihara ◽

Yasuyuki Igarashi

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Family Members ◽

Fatty Acyl ◽

Ceramide Synthase ◽

Broad Substrate Specificity ◽

Acid Protein ◽

Amino Acid Protein

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

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First characterization of an archaeal amino acid racemase with broad substrate specificity from the hyperthermophile Pyrococcus horikoshii OT-3

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2017.02.004 ◽

2017 ◽

Vol 124 (1) ◽

pp. 23-27 ◽

Cited By ~ 3

Author(s):

Ryushi Kawakami ◽

Haruhiko Sakuraba ◽

Taketo Ohmori ◽

Toshihisa Ohshima

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Pyrococcus Horikoshii ◽

Broad Substrate Specificity ◽

Amino Acid Racemase

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Identification of Amino Acid Residues of the Retroviral Aspartic Proteinases Important for Substrate Specificity and Catalytic Efficiency

Aspartic Proteinases - Advances in Experimental Medicine and Biology ◽

10.1007/978-1-4615-1871-6_52 ◽

1995 ◽

pp. 399-406 ◽

Cited By ~ 1

Author(s):

C. E. Cameron ◽

H. Burstein ◽

D. Bizub-Bender ◽

T. Ridky ◽

I. T. Weber ◽

...

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Catalytic Efficiency ◽

Amino Acid Residues ◽

Aspartic Proteinases

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Prediction of amino acid residues participated in substrate recognition by cytochrome P450 subfamilies with broad substrate specificity

Journal of Molecular Recognition ◽

10.1002/jmr.2251 ◽

2013 ◽

Vol 26 (2) ◽

pp. 86-91 ◽

Cited By ~ 5

Author(s):

Maria S. Zharkova ◽

Boris N. Sobolev ◽

Nina Yu. Oparina ◽

Alexander V. Veselovsky ◽

Alexander I. Archakov

Keyword(s):

Cytochrome P450 ◽

Amino Acid ◽

Substrate Specificity ◽

Substrate Recognition ◽

Amino Acid Residues ◽

Broad Substrate Specificity

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Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l-Threonine Accumulation by Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.68.7.3321-3327.2002 ◽

2002 ◽

Vol 68 (7) ◽

pp. 3321-3327 ◽

Cited By ~ 61

Author(s):

Petra Simic ◽

Juliane Willuhn ◽

Hermann Sahm ◽

Lothar Eggeling

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Corynebacterium Glutamicum ◽

Serine Hydroxymethyltransferase ◽

Acid Formation ◽

Initial Activity ◽

Intracellular Degradation ◽

Cleavage Activity ◽

Substrate Reduction

ABSTRACT l-Threonine can be made by the amino acid-producing bacterium Corynebacterium glutamicum. However, in the course of this process, some of the l-threonine is degraded to glycine. We detected an aldole cleavage activity of l-threonine in crude extracts with an activity of 2.2 nmol min−1 (mg of protein)−1. In order to discover the molecular reason for this activity, we cloned glyA, encoding serine hydroxymethyltransferase (SHMT). By using affinity-tagged glyA, SHMT was isolated and its substrate specificity was determined. The aldole cleavage activity of purified SHMT with l-threonine as the substrate was 1.3 μmol min−1 (mg of protein)−1, which was 4% of that with l-serine as substrate. Reduction of SHMT activity in vivo was obtained by placing the essential glyA gene in the chromosome under the control of P tac , making glyA expression isopropylthiogalactopyranoside dependent. In this way, the SHMT activity in an l-threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while l-threonine was simultaneously increased by 49%. The intracellular availability of l-threonine to aldole cleavage was also reduced by overexpressing the l-threonine exporter thrE. In C. glutamicum DR-17, which overexpresses thrE, accumulation of 67 mM instead of 49 mM l-threonine was obtained. This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.

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