Structural basis of the selectivity of GenN, an aminoglycoside N-methyltransferase involved in gentamicin biosynthesis

Priscila dos Santos Bury, Fanglu Huang, Sicong Li, Yuhui Sun, Peter F. Leadlay, and Marcio Vinicius Bertacine Dia*

ACS Chemical Biology 2017, 12(11):2779-2787

Epub Date: 9 October 2017

DOI: 10.1021/acschembio.7b00466


Gentamicinsare heavily methylated, clinically valuable pseudotrisaccharide antibiotics produced by Micromonospora echinospora. GenN has been characterized as an S-adenosyl-L-methionine-dependent methyltransferase with low sequence similarity to other enzymes. It isresponsible for the 3″-N-methylation of 3″-dehydro-3″-amino-gentamicin A2, an essential modification of ring III in the biosynthetic pathway to the gentamicin C complex. Purified recombinant GenN also efficiently catalyzes 3″-N-methylation of related aminoglycosides kanamycin B and tobramycin, which both contain an additional hydroxymethyl group at the C5″ position in ring III. We have obtained eight cocrystal structures of GenN, at a resolution of 2.2 Å orbetter, including the binary complex of GenN and S-adenosyl-l-homocysteine (SAH) and the ternary complexes of GenN, SAH, and several aminoglycosides. The GenN structure reveals several features not observed in any other N-methyltransferase that fit it for its role in gentamicin biosynthesis. These include a novel N-terminal domain that might be involved in protein:protein interaction with upstream enzymes of the gentamicin X2 biosynthesis and twolong loops that are involved in aminoglycoside substrate recognition. In addition, the analysis of structures of GenN in complex with different ligands, supported by the results of active site mutagenesis, has allowed us to proposea catalytic mechanism and has revealed the structural basis for the surprising ability of native GenN to act on these alternative substrates.

Functional analysis of cytochrome P450s involved in streptovaricin biosynthesis and generation of anti-MRSA analogues

Yuanzhen Liu, Xu Chen, Zhengyuan Li, Wei Xu, Weixin Tao, Jie Wu, Jian Yang, Zixin Deng, and Yuhui Sun*

ACS Chemical Biology 2017, 12(10):2589-2597

Epub Date: 7 September 2017

DOI: 10.1021/acschembio.7b00467


Thestreptovaricins, chemically related to the rifamycins, are highly effective antibacterial agents particularly against mycobacteria. Herein, abioassay-guided investigation of Streptomyces spectabilis CCTCCM2017417 has led to the characterization of streptovaricins as potent compounds against methicillin-resistant Staphylococcus aureus (MRSA). Targetedin-frame deletion of five cytochrome P450 genes (stvP1-P5) resulted in the identification of four new streptovaricin analogues, and revealed the functions of these genes as follows: stvP1, stvP4 and stvP5 are responsible for the hydroxylation of C-20, Me-28 and C-24, respectively; stvP2 is possibly involved in formation of the methylenedioxy bridge, and stvP3, a conserved gene found in the biosynthetic cluster for naphthalenic ansamycins, might be related to the formation of naphthalene ring. Biochemical verification of the hydroxylase activity of StvP1, StvP4 and StvP5 was performed, and StvP1 showed unexpected biocatalytic specificity and promiscuity. More importantly, anti-MRSA studies of streptovaricins and derivatives revealed significant structure-activity relationships (SARs). This work provides meaning fulinformation on the SARs of streptovaricins, and demonstrates the utility of the engineering of streptovaricins to yield novel anti-MRSA molecules.

An iterative module in the azalomycin F polyketide synthase contains a switchable enoylreductase domain

Wei Xu#, Guifa Zhai#, Yuanzhen Liu, Yuan Li, Yanrong Shi, Kui Hong, Hui Hong, Peter F. Leadlay, Zixin Deng and Yuhui Sun*

Angewandte Chemie International Edition 2017, 56(20):5503-5506

Epub Date: 18 April 2017

DOI: 10.1002/anie.201701220


Detailed analysis of the modular Type I polyketide synthase (PKS) involved in biosynthesis of the marginolactone azalomycin F in mangrove Streptomyces sp. 211726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of a PKS mutant specifically inactivated in the dehydratase domain of extension module 1 has proved that this module catalyses two successive elongations with different outcomes. Strikingly, the enoylreductase domain of this module is apparently "toggled" on and off: it functions in only the second of these two cycles. This novel mechanism expands the paradigm of PKS assembly-line catalysis and may explain examples of apparent non-colinearity in other modular PKS systems.

The polyketide backbone of thiolactomycin is assembled by an unusual iterative polyketide synthase

Marie E. Yurkovich, Robert Jenkins, Yuhui Sun, Manuela Tosin*, and Peter F. Leadlay*

Chemical Communications 2017, 53(13):2182-2185

Epub Date: 26 January 2017

DOI: 10.1039/c6cc09934c


Following the in vivo investigation of thiotetronate assembly in Lentzea sp. and in S. thiolactonus NRRL 15439 (Havemann et al., Chem. Commun., 2017, DOI: 10.1039/c6cc09933e), the minimal set of genes required for thiolactomycin production was determined through heterologous expression and the mechanism for polyketide assembly was established in vitro through incubation of recombinant TlmB with its substrates in the presence of either nonhydrolysable or hydrolysable chemical probes. The results presented here constitute unequivocal evidence of enzymatic processing by an unusual iterative polyketide synthase.

Chemical probing of thiotetronate bio-assembly

Judith Havemann, Marie E. Yurkovich, Robert Jenkins, Sophia Harringer, Weixin Tao, Shishi Wen, Yuhui Sun, Peter F. Leadlay, and Manuela Tosin*

Chemical Communications 2017, 53(11):1912-1915

Epub Date: 25 January 2017

DOI: 10.1039/c6cc09933e


Chemical ‘chain termination’ probes were utilised for the investigation of thiotetronate antibiotic biosynthesis in the filamentous bacteria Lentzea sp. and Streptomyces thiolactonus NRRL 15439. The use of these tools led to the capture of biosynthetic intermediates involved in the thiotetronate polyketide backbone assembly, providing first insights into substrate specificity and in vivo intermediate processing by unusual iterative synthases.


New era of gene editing driven by CRISPR-Cas9


Binbin Xiong, HuZeng, Yunkun Liu, and YuhuiSun*

微生物学通报 2017, 44(1):175-185

Microbiology China 2017, 44(1):175-185 (Chinese)

Published online: 13 April 2016



在自然界生物长期的进化过程中,细菌和古细菌演化出了一种适应性免疫系统用以抵御外源病毒与质粒的入侵,该系统由成簇规律间隔的短回文重复序列与相关基因组成,称之为CRISPR-Cas。近年来,这一领域突飞猛进,如今已经发展成为一种功能强大的基因编辑工具并在生物学及其相关领域得到广泛应用。本文重点综述了近年来CRISPR-Cas9 系统在基因编辑、基因调节以及作为体外工具酶和特异性等方面的若干前沿进展

During long-term evolution of life in nature, bacteria and archaea have developed anadaptive defense systemcalled clustered regularly interspaced short palindromic repeat sequences and CRISPR-associated genes (CRISPR-Cas) that protect organisms from invading viruses and plasmids. It has been recently used as a powerful gene-editing tool in a wide variety of important organisms. In this review, we summarize recent progresses on CRISPR-Cas9 system and its applications to gene editing, gene regulation, and in vitro DNA manipulations used as a programmable molecular clone tool.