l-Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family

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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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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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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Functional and structural characterization of IdnL7, an adenylation enzyme involved in incednine biosynthesis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19002863 ◽

2019 ◽

Vol 75 (4) ◽

pp. 299-306

Author(s):

Jolanta Cieślak ◽

Akimasa Miyanaga ◽

Makoto Takaishi ◽

Fumitaka Kudo ◽

Tadashi Eguchi

Keyword(s):

Amino Acids ◽

Substrate Specificity ◽

Natural Product ◽

Structural Characterization ◽

Biochemical Analysis ◽

Natural Product Biosynthesis ◽

Broad Substrate Specificity ◽

Selective Incorporation ◽

Polyketide Antibiotic

Adenylation enzymes play an important role in the selective incorporation of the cognate carboxylate substrates in natural product biosynthesis. Here, the biochemical and structural characterization of the adenylation enzyme IdnL7, which is involved in the biosynthesis of the macrolactam polyketide antibiotic incednine, is reported. Biochemical analysis showed that IdnL7 selects and activates several small amino acids. The structure of IdnL7 in complex with an L-alanyl-adenylate intermediate mimic, 5′-O-[N-(L-alanyl)sulfamoyl]adenosine, was determined at 2.1 Å resolution. The structure of IdnL7 explains the broad substrate specificity of IdnL7 towards small L-amino acids.

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