Tabular analysis of polyketide synthase domain active site residues for the PKZILLAs

In brief, the presence or absence of expected conserved enzyme active site residues within domains of the PKZILLAs, were matched to the polyketide ketosynthase (KS), acyl carrier protein (ACP), dehydratase (DH), ketoreductase (KR), flavin oxygenase (FLX), and enoyl (ER) domains, using either multiple sequence alignment liftover of active site location & residue as defined by InterProScan annotations, or, locations & residues defined as active site residues from literature sources. ./KS_ALL represents the scripts and source files used to generate the analyses. See run.sh , within. Post_process_KS-ALL_analysis.ipynb represents the Jupyter notebook used to process the raw analyses into the presented tabular (.xlsx) form. See Manuscript Materials and Methods for further details. Summary of the active side residue results: KS:All scored as active on the basis of catalytic cysteine. The below KS domains are scored as KS0, on the basis of variation of the histidine in the HGTGT motif: PKZILLA-1__KS14 QGLGS PKZILLA-1__KS19 QGLGS PKZILLA-1__KS22 QGLGS PKZILLA-1__KS25 QGLGS PKZILLA-1__KS28 QGLGS PKZILLA-1__KS31 GANGS PKZILLA-1__KS34 QGLGS PKZILLA-2__KS3 QGLGS PKZILLA-2__KS6 QGLGS PKZILLA-2__KS9 QGLGS PKZILLA-2__KS11 QGLGS PKZILLA-2__KS12 QGLGS PKZILLA-3__KS6 AANGS ACP:All scored as active (PPant prosthetic group attachment residue present) DH:All scored as active, except possibly:PKZILLA-3__DH9 ER:Active site residues not defined in InterProScan or literature sources. FLX:Active site residues not defined in InterProScan or literature sources. KR: KR domains come in two flavors: the canonical "full-length" contiguous domain, and the non-canonical split domain with two subdomains.KRs are 2 reciprocally similar subdomains - where the N-terminal subdomain "KRn" has a structural role, and the C-terminal subdomain "KRc" has the catalytic role. Typically, the two KR subdomains are contiguous. However, it's precedendented (Porcine FAS) that these two KRn and KRc subdomains can become split with an ER in the middle. In the PKZILLAs, this can be observed for the KR-ER-KR in Module 28, it's actually KRn-ER-KRc. The first KRn-subdomain (KR26) is missing the catalytic residues (as are all of the KRn domains), while the second KRc-subdomain (KR27) has all of them. This split KR domain structure seems to be not that uncommon in the PKZILLAs, and the intervening domain is not limited to being an ER. The KR.xlsx excel summarizes the status of the KRs, as active or inactive, and full-length (f), n-terminal subdomain (n), or c-terminal subdomain (c). The below KR domains are scored as inactive: PKZILLA-1__KR26PKZILLA-1__KR29PKZILLA-1__KR31PKZILLA-2__KR6PKZILLA-2__KR11PKZILLA-2__KR20 Summary of the domain phylogenetics results: Present in separate Zenodo item: https://doi.org/10.5281/zenodo.10152639简言之，我们将PKZILLA家族结构域中预期的保守酶活性位点残基的存在与否，与聚酮合酶酮合成酶（polyketide ketosynthase, KS）、酰基载体蛋白（acyl carrier protein, ACP）、脱水酶（dehydratase, DH）、酮还原酶（ketoreductase, KR）、黄素氧化酶（flavin oxygenase, FLX）以及烯酰还原酶（enoyl reductase, ER）结构域进行匹配；匹配依据为InterProScan注释所定义的活性位点位置与残基的多序列比对映射，或是文献来源中界定的活性位点位置与残基。 KS_ALL目录包含用于生成本次分析的脚本与源文件，详见其中的run.sh脚本。 Post_process_KS-ALL_analysis.ipynb为Jupyter Notebook文件，用于将原始分析结果处理为本文呈现的表格形式（.xlsx格式）。 更多细节请参阅论文的材料与方法部分。 活性位点残基分析结果总结： KS结构域：所有结构域均基于催化半胱氨酸被判定为具有活性。以下KS结构域因HGTGT基序中的组氨酸发生变异，被归类为KS0型： PKZILLA-1__KS14 QGLGS PKZILLA-1__KS19 QGLGS PKZILLA-1__KS22 QGLGS PKZILLA-1__KS25 QGLGS PKZILLA-1__KS28 QGLGS PKZILLA-1__KS31 GANGS PKZILLA-1__KS34 QGLGS PKZILLA-2__KS3 QGLGS PKZILLA-2__KS6 QGLGS PKZILLA-2__KS9 QGLGS PKZILLA-2__KS11 QGLGS PKZILLA-2__KS12 QGLGS PKZILLA-3__KS6 AANGS ACP结构域：所有结构域均被判定为具有活性（存在磷酸泛酰巯基乙胺（phosphopantetheine, PPant）辅基附着残基）。 DH结构域：除PKZILLA-3__DH9外，所有结构域均被判定为具有活性。 ER结构域：InterProScan注释与文献均未界定其活性位点残基。 FLX结构域：InterProScan注释与文献均未界定其活性位点残基。 KR结构域： KR结构域存在两种类型：经典的"全长"连续结构域，以及具有两个亚结构域的非经典分裂型结构域。KR由两个互为同源的亚结构域组成，其中N端亚结构域"KRn"仅发挥结构作用，而C端亚结构域"KRc"承担催化功能。 通常情况下，这两个KR亚结构域是连续的。不过，已有研究先例（以猪脂肪酸合酶（Porcine FAS）为例）表明，这两个KRn与KRc亚结构域可被中间插入的ER结构域分隔。在PKZILLA家族中，模块28中的KR-ER-KR结构即为这类分裂型结构，实际为KRn-ER-KRc。首个KRn亚结构域（KR26）缺失催化残基（所有KRn亚结构域均存在此情况），而第二个KRc亚结构域（KR27）则包含全部催化残基。这类分裂型KR结构域在PKZILLA家族中并不罕见，且中间插入的结构域并非仅局限于ER。 KR.xlsx文件汇总了所有KR结构域的状态：包括活性/非活性，以及全长（f）、N端亚结构域（n）或C端亚结构域（c）类型。以下KR结构域被判定为非活性： PKZILLA-1__KR26 PKZILLA-1__KR29 PKZILLA-1__KR31 PKZILLA-2__KR6 PKZILLA-2__KR11 PKZILLA-2__KR20 结构域系统发育分析结果总结： 相关数据已上传至独立的Zenodo资源库：https://doi.org/10.5281/zenodo.10152639