Crystallization and preliminary X-ray diffraction analysis of a putative two-domain-type laccase from a metagenome

Kakemoto, Hirofumi; Miyazaki, Kentaro; Higuchi, Yoshiki

doi:10.1107/s1744309109002462

Cited by 15 publications

(19 citation statements)

References 24 publications

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“…mgLAC was prepared as previously described [18]. Enzyme activities were measured from the decrease in the concentration of dissolved molecular oxygen at 25 °C in McIlvaine buffer (pH 4.5 for ABTS and 8.5 for others), containing 1 mM substrate on a PreSens oxygen meter (Microx TX3).…”

Section: Methodsmentioning

confidence: 99%

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X‐ray structure of a two‐domain type laccase: A missing link in the evolution of multi‐copper proteins

2009

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a b s t r a c tA multi-copper protein with two cupredoxin-like domains was identified from our in-house metagenomic database. The recombinant protein, mgLAC, contained four copper ions/subunits, oxidized various phenolic and non-phenolic substrates, and had spectroscopic properties similar to common laccases. X-ray structure analysis revealed a homotrimeric architecture for this enzyme, which resembles nitrite reductase (NIR). However, a difference in copper coordination was found at the domain interface. mgLAC contains a T2/T3 tri-nuclear copper cluster at this site, whereas a mononuclear T2 copper occupies this position in NIR. The trimer is thus an essential part of the architecture of two-domain multi-copper proteins, and mgLAC may be an evolutionary precursor of NIR.

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Section: Methodsmentioning

confidence: 99%

“…Crystallization and preliminary X‐ray crystallographic results are described as previously noted [18]. The crystal belongs to space group P 2 1 2 1 2 1 , with unit‐cell parameters of a = 74.67, b = 100.95, and c = 124.11 Å.…”

Section: Methodsmentioning

confidence: 99%

X‐ray structure of a two‐domain type laccase: A missing link in the evolution of multi‐copper proteins

2009

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“…The small molecular weight laccases identified to date are SLAC (small laccase from Streptomyces coelicolor (15)), EpoA from Streptomyces griseus (14), and mgLAC (laccase from metagenome of sludge) (44). It has stimulated efforts to discover new types of laccases.…”

Section: Two-domain Type Mco (2dmco)mentioning

confidence: 99%

Structure and molecular evolution of multicopper blue proteins

Kakemoto¹,

Higuchi²

2010

BioMolecular Concepts

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The multicopper blue protein family, which contains cupredoxin-like domains as a structural unit, is one of the most diverse groups of proteins. This protein family is divided into two functionally different types of enzymes: multicopper oxidase and nitrite reductase. Multicopper oxidase catalyzes the oxidation of the substrate and then reduces dioxygen. The structures of many multicopper oxidases are already known, and until recently they were classified into two main groups: the three- and six-domain types. Both function as monomers and have three spectroscopically different copper sites: Types I (blue), II, and III (tri-nuclear). Nitrite reductase is a closely related protein that contains Types I and II (mono-nuclear) coppers but reduces nitrite instead of dioxygen. Nitrite reductase, which consists of two domains, forms a homotrimer. Multicopper oxidase and nitrite reductase share similar structural architectures and also contain Type I copper. Therefore, it is proposed that they have a common ancestor protein. Recently, some two-domain type multicopper oxidases have been found and their crystal structures have been determined. They have a trimeric quaternary structure and contain an active site at the molecular interface such as nitrite reductase. These results support previous hypotheses and provide an insight into the molecular evolution of multicopper blue proteins.

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“…However, since more than 99% of microbes in the environment are not easily cultivated (34), metagenomic approaches may circumvent culture-based investigations and comprehensively capture a diverse array of target gene families (14). Novel biocatalysis-relevant enzymes originating from environmental microbial communities have been discovered using a metagenomic approach (22, 51). For example, a gene encoding a novel MOC with laccase activity was identified through the activity-based functional screening of a metagenomic library from mangrove soil (51), and a novel MOC was also identified through the activity screening of a metagenome expression library from bovine rumen microflora (3).…”

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confidence: 99%

A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach

Kimura

Kamagata

2016

Microb. Environ.

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A gene coding for a multicopper oxidase (BopA) was identified through the screening of a metagenomic library constructed from wastewater treatment activated sludge. The recombinant BopA protein produced in Escherichia coli exhibited oxidation activity toward 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of copper, suggesting that BopA is laccase. A bioinformatic analysis of the bopA gene sequence indicated that it has a phylogenetically bacterial origin, possibly derived from a bacterium within the phylum Deinococcus-Thermus. Purified BopA exhibited maximum activity at pH 7.5 with bilirubin as its substrate and was found to be active over a markedly broad pH range from 6 to 11. It also showed notable thermostability; its activity remained intact even after a heat treatment at 90°C for 60 min. This enzyme is a thermostable-bilirubin oxidase that exhibits markedly higher thermostability than that previously reported for laccases.

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Crystallization and preliminary X-ray diffraction analysis of a putative two-domain-type laccase from a metagenome

Cited by 15 publications

References 24 publications

X‐ray structure of a two‐domain type laccase: A missing link in the evolution of multi‐copper proteins

X‐ray structure of a two‐domain type laccase: A missing link in the evolution of multi‐copper proteins

Structure and molecular evolution of multicopper blue proteins

A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach

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